Androgen treatment in females

ABSTRACT

Method of improving ovarian reserve in a human female with diminished ovarian reserve as measured by the female&#39;s anti-Müllerian hormone level. The method may include evaluating a first anti-Müllerian hormone level of the female, administering an androgen, such as dehydroepiandrosterone or testosterone, to the female for at least about one month, and then evaluating a second anti-Müllerian hormone level of the female. Change in the anti-Müllerian hormone level is indicative of change in the ovarian reserve, e.g., when the second anti-Müllerian hormone level is greater than the first anti-Müllerian hormone level, the ovarian reserve has improved. The androgen administration may continue until the second anti-Müllerian hormone level is greater than the first anti-Müllerian hormone level by a desired percentage or amount.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/575,426 filed Oct. 7, 2009, which is a continuation-in-part of U.S.patent application Ser. No. 11/269,310, filed Nov. 8, 2005, and U.S.patent application Ser. No. 12/123,877, filed May 20, 2008, all of whichare incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of reproductive technology.

2. Description of the Related Art

The application of assisted reproductive technology has revolutionizedthe treatment of all forms of infertility. The most common assistedreproductive technology is in vitro fertilization (IVF), in which awoman's eggs are harvested and fertilized with a man's sperm in alaboratory. Embryos grown from the sperm and eggs are then chosen to betransferred into the woman's uterus. Assisted reproductive technology inwomen depends on ovarian stimulation and concurrent multiple oocytedevelopment, induced by exogenous gonadotropins.

Infertile women are often treated with gonadotropin treatments such asgonadotropin-releasing hormone (GnRH) flare protocols. Estrogenpre-treatment with concomitant growth hormone (GH) treatment issometimes used in an effort to try and amplify intra-ovarianinsulin-like growth factor-I (IGF-I) paracrine effect, which isexpressed by granulosa cells and enhances gonadotropin action. However,the clinical utility of combined GH/ovarian stimulation is limited andresponses are not dramatic.

Dehydroepiandrosterone (DHEA) is secreted by the adrenal cortex, centralnervous system and the ovarian theca cells and is converted inperipheral tissue to more active forms of androgen or estrogen. DHEAsecretion during childhood is minimal but it increases at adrenarche andpeaks around age 25, the age of maximum fertility, only to reach a nadirafter age 60. There is evidence to support use of exogenous DHEA toincrease ovulation stimulation in older women who respond poorly togonadotropin treatments.

Women with diminished ovarian function have decreased egg production andthe eggs that are produced usually are of a poor quality. Further, womenwith diminished ovarian function tend to encounter difficulty becomingpregnant with or without IVF and experience long time periods toconception.

Even when these women do achieve a pregnancy, the rate of a possiblemiscarriage increases. A large majority of approximately 80 percent ofspontaneous pregnancy loss is the consequence of chromosomalabnormalities. As women get older and their ovarian functionprogressively declines, miscarriage rates rise because of increasinganeuploidy.

Women with diminished ovarian function have largely been considered tobe untreatable.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method of improving ovarianreserve in a human female with diminished ovarian reserve as measured bythe female's anti-Müllerian hormone level. The method may includeevaluating a first anti-Müllerian hormone level of the female,administering an androgen, such as dehydroepiandrosterone ortestosterone, to the female for at least about one month based on theevaluation of the first anti-Müllerian hormone level, and evaluating asecond anti-Müllerian hormone level of the female, wherein improvementof the ovarian reserve is indicated when the second anti-Müllerianhormone level is greater than the first anti-Müllerian hormone level.

The present invention also is directed to a method of evaluating theeffect of an androgen, such as dehydroepiandrosterone or testosterone,in a human female with diminished ovarian reserve. The method mayinclude evaluating a first anti-Müllerian hormone level of the female,administering dehydroepiandrosterone to the female for at least aboutone month based on the evaluation of the first anti-Müllerian hormonelevel, evaluating a second anti-Müllerian hormone level of the female,and comparing the second anti-Müllerian hormone level to the firstanti-Müllerian hormone level. This comparison provides an indication ofthe effect of the androgen on the ovarian reserve.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table showing improved ovulation induction with DHEA.

FIG. 2 is a graph showing an increase in the number of fertilizedoocytes resulting from oocytes harvested from women with DHEA treatment.

FIG. 3 is a graph showing an increase in the number of fertilizedoocytes resulting from oocytes harvested from women with at least 4weeks of DHEA treatment.

FIG. 4 is a graph showing an increase in the number of day three embryosresulting from oocytes harvested from women with at least 4 weeks ofDHEA treatment.

FIG. 5 is a chart showing chemical pathways of adrenal function.

FIG. 6 is a graph showing cumulative pregnancy rate of time from initialvisit to clinical pregnancy or censor by DHEA for women with prematureovarian aging.

FIG. 7 is a graph showing cumulative pregnancy rate of time from initialvisit to clinical pregnancy or censor by DHEA for women with diminishedovarian reserve.

FIG. 8 is a graph showing a comparison of miscarriage rates between DHEAtreated infertility patients and 2004 national US IVF data.

FIG. 9 is a graph showing a cross-sectional evaluation of AMH levels incorrelation to time from DHEA initiation.

FIG. 10 is a graph showing levels over time from DHEA initiation inwomen who did and did not conceive.

DETAILED DESCRIPTION OF THE INVENTION

When attempting in vitro fertilization (IVF), older women produce fewoocytes and yield few normal embryos, even when exposed to maximalgonadotropin stimulation. The decreased ability of older women torespond to ovulation inducing medications is evidence that ovarianreserve declines with age. Even with IVF cycles, older women produce fewoocytes and yield few normal embryos when exposed to maximalgonadotropin stimulation. This change in ovarian responsiveness is knownas diminished ovarian reserve or diminished ovarian function.

To improve the number of eggs, the quality of eggs, the number ofembryos, the quality of the embryos, spontaneous pregnancy rates, IVFpregnancy rates, cumulative pregnancy rates and time to conception, toreduce the miscarriage rates, and to increase the male/female birthratio, DHEA is administered for at least two months to a human female ina therapeutically effective amount. Preferably, the human female is apremenopausal human female. The human female may have diminished ovarianreserve. DHEA may be administered to a human female at a dose of betweenabout 50 mg/day and about 100 mg/day, preferably between about 60 mg/dayand about 80 mg/day, and in one study about 75 mg/day. Further, DHEA maybe administered in a time-release formulation, over the course of theday, or in a single dose. For example, the about 75 mg/day could beadministered in a single dose of 75 mg or could be administered as 25 mgthree times throughout the day. DHEA is preferably administered orally,although DHEA may be administered or delivered via other methods, suchas, but not limited to, intravenously and/or topically. DHEA has astatistically significant effect on the above-mentioned factors afterabout 2 months of use, but its effect may continue to increase to aboutfour months or about 16 weeks, preferably about four consecutive monthsor about 16 consecutive weeks, and further may continue past four monthsof use.

The effects of DHEA increase over time, and may reach peaks afterapproximately four to five months of supplementation. It is suggestedthat peaks may occur at four to five months because this time period issimilar to the time period of a complete follicular recruitment cycle.Further, the effect of DHEA is suggested to reduce chromosomalabnormalities and thus substantially decreasing miscarriage rates inhuman females.

I. Improvements in Ovulation

Treatments with an androgen, alone or in conjunction with otherhormones, increase a woman's response to ovulation induction, measuredin both oocyte and embryo yield. Androgens may be, for example,dehydroepiandrosterone (DHEA) or testosterone. DHEA treatment may be anadjunct to ovulation induction. DHEA taken orally for at least about onemonth, preferably for about four months, before optionally initiatinggonadotropin treatment, may prepare the ovaries for gonadotropinstimulation. A large response may be obtainable by combininggonadotropins and DHEA in treatment for at least about a four monthperiod before an IVF cycle.

Young ovaries are characterized by large numbers of antral follicles anda low rate of atresia. In contrast, older ovaries have few antralfollicles, high rates of atresia and exhibit increasing “resistance” toovulation induction. Older women have decreased oocyte quantity andquality, produce fewer high quality embryos and have lower implantationand pregnancy rates. Most follicular atresia occurs after the primordialfollicle resumes growth but before it is gonadotropin responsive enoughfor recruitment. An induced delay in onset of atresia may salvagefollicles for possible ovulation. Interestingly, such an “arrest” of theatretic process has been noted among anovulatory women with polycysticovary syndrome (PCO). For these women follicles remain steroidogenicalycompetent and show evidence of increased aromatase activity compared tolike-sized follicles from normal ovaries. Follicular hypersecretion ofDHEA, which is typical of PCO, is associated with increased aromataseactivity. The increased yield of oocytes and embryos experienced bypatients undergoing DHEA treatment may correspond to this underlyingphysiological process.

II. Improvements to Cumulative Embryo Score

DHEA use beneficially effects oocyte and embryo quality. The observationthat DHEA treatment is associated with improved cumulative embryo scoresinfers that such treatment leads to improved embryo and egg quality.This suggestion is further supported by strong trends towards improvedeuploidy in embryos and improved pregnancy rates.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA has a statisticallysignificant effect on cumulative embryo score after about 2 months ofadministration, but its effect may continue to increase to about fourmonths, or about 16 weeks, and further may continue past four months ofuse.

Cumulative embryo score is determined by scoring day 3 embryos andmultiplying the number of cells in the embryo by the embryo grade.Embryo grade is a judgment of the embryologist on embryo quality from 1to 5. Most good embryos are scored 4, with 5 reserved for exceptionalembryos. The grade is based on the uniformity of the cells, the colorand consistency of the cytoplasm, and the amount of fragmentation.Normal embryos are less than 5% fragmented. A woman with three eightcell embryos each with a grade of four would have a cumulative embryosscore of 96, the product of 3×8×4.

A cumulative embryo score for women prior to DHEA use may have beenabout 34. A cumulative embryo score after DHEA use of at least aboutfour consecutive months may be at least about 90, preferably at leastabout 95, and in one study at least about 98. The increase in cumulativeembryo score may be at least about 56, preferably at least about 60, andin one study about 64. The difference in the cumulative embryo scoreprior to DHEA use and the cumulative embryo score after DHEA use isstatistically significant, p<0.001. The mean increase in embryo scorewas about 57+/−14.7 after about 16.1 weeks of DHEA administration. Assuch, DHEA treatment significantly improves the cumulative embryo score.

III. Increase in the Number of Fertilized Oocytes

DHEA treatment significantly increased the number of fertilized oocytesproduced by women. DHEA treatment includes administering a dose ofbetween about 50 mg/day and about 100 mg/day, preferably between about60 mg/day and about 80 mg/day, and in one study about 75 mg/day to ahuman female. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect on the number of fertilized oocytes after about 4 consecutiveweeks. However, DHEA has a significant effect on the number offertilized oocytes after about 8 weeks or about 2 months ofadministration, and its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after about atleast 16 weeks or about at least 4 months of administration.

The number of fertilized oocytes produced by women significantlyincreased after at least about 4 months of consecutive DHEA treatment in12 women, even though slight improvements were shown after at leastabout four weeks of consecutive DHEA treatment, as shown in FIG. 3. Asshown in FIG. 3, paired comparisons of fertilized oocytes from womenhaving less than about four consecutive weeks of DHEA treatment to thesame women having at least about four consecutive weeks of DHEAtreatment showed an increase of about 2 fertilized oocytes, or a medianincrease of about 2.5 fertilized oocytes. The number of fertilizedoocytes may show more significant increase after at least about 4 monthsof DHEA treatment, and may show maximal increase after at least abouteight months of DHEA treatment.

IV. Increase in the Number of Day 3 Embryos

DHEA treatment significantly increased the number of day 3 embryosproduced by women. DHEA treatment includes administering a dose ofbetween about 50 mg/day and about 100 mg/day, preferably between about60 mg/day and about 80 mg/day, and in one study about 75 mg/day to ahuman female. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect of day 3 embryos after about 4 consecutive weeks. However, DHEAhas a significant effect after about 8 weeks or about 2 months ofadministration, but its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after about atleast 16 weeks or about at least 4 months of administration.

The number of day 3 embryos produced by women also may significantlyincrease after at least about four months of consecutive DHEA treatmentin 12 women, even though slight increases may be shown after at leastabout 4 weeks of DHEA treatment, as shown in FIG. 4. All of the day 3embryos included in the study were normal based on their appearance andon the number of cells, i.e. at least four cells. Paired comparisons offertilized oocytes from women having less than about four consecutiveweeks of DHEA treatment to the same women having at least about fourconsecutive weeks of DHEA treatment may show an increase of about 1 day3 embryo, and in the study summarized in FIG. 4, an increase of about 2day 3 embryos. While the number of day 3 embryos produced slightlyincreased after at least 4 weeks of DHEA treatment, more significantincrease occurs after at least about 4 months of DHEA treatment, andmaximal increase may occur after at least about eight months of DHEAtreatment.

V. Increase in the Number of Euploid Oocytes

DHEA may improve the number of euploid embryos and embryo transfers inwomen with diminished ovarian reserve (DOR). Pretreatment with DHEA, forat least about one month, preferably at least about four months, inwomen may increase oocyte and embryo quantity, egg and embryo quality,cumulative pregnancy rates, pregnancy rates with IVF and time topregnancy.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a significant effect afterabout 8 weeks or about 2 months of administration, but its effect maycontinue to increase to about four months, and further may continue pastfour months of use. Specifically, DHEA treatment has a statisticallysignificant effect after about at least 16 weeks or about at least 4months of administration.

The prevalence of aneuploidy in embryos, produced through IVF, from 27consecutive IVF cycles in women with DOR who also had undergonepreimplantation genetic diagnosis (PGD) was evaluated. Amongst thosecycles, 19 had entered IVF without DHEA treatment and eight had receivedDHEA supplementation for at least four weeks prior to IVF start.

DHEA treatment may result in higher oocyte numbers (10.4±7.3 vs.8.5±4.6) increasing from about 8.5 to about 10.4. A significantly largernumber of DHEA treated IVF cycles ( 8/8, 100%) had at least one euploidembryo for transfer than in untreated cycles ( 10/19, 52.6%; Likelihoodratio, p=0.004; Fisher's Exact Test, p=0.026). Neither absolute numbersof euploid embryos after DHEA nor percentages of euploid embryosdiffered significantly in this case, however, between untreated andtreated patients.

As women age, there is a substantial decline in euploidy rates inembryos produced. Thus, the increase in euploidy results in older womenis dramatic evidence of the effectiveness of DHEA in improving embryoquality, and pretreatment with DHEA of women with DOR may significantlyincrease their chances for the transfer of at least one euploid embryo.

VI. Improvements to Ovarian Function

DHEA may have beneficial effects on ovarian function and oocyte andembryo quality. DHEA substitution may rejuvenate certain aspects ofovarian function in older ovaries. Since DHEA declines with age to avery significant degree, intraovarian DHEA deficiency may be causallyrelated to the ovarian aging process.

FIG. 5 shows the pathways for normal adrenal function. As shown in FIG.5, the adrenal enzyme 17,20-desmolase may be responsible for theconversion of 17-hydroxy pregnenolone into DHEA (and the conversion of17-hydroxyprogesterone into androstenedione) which, based on thetwo-cell two-gonadotropin theory, may serve in the ovary as a precursorsubstrate for estradiol and androgens. A patient (Patient B), describedfurther in Example 5 herein, with abnormal 17,20-desmolase (P450c17)function may have a hormone profile characterized by persistently lowDHEA, androstenedione, testosterone and estradiol levels, but normalaldosterone and cortisol levels. Patient B exhibited some of theclassical signs of prematurely aging ovaries which include ovarianresistance to stimulation, poor egg and embryo quality and prematurelyelevated FSH levels.

The decrease in DHEA levels with advancing female age may be an inherentpart of the ovarian aging process and may, at least in part, and on atemporary basis, be reversed by external DHEA substitution. This casedemonstrates that low DHEA levels are, indeed, associated with all theclassical signs of (prematurely) aging ovaries. While association doesnot necessarily suggest causation, the observed sequence of events inthis patient supports the notion that low DHEA levels adversely affectovarian function.

Patient B was initially thought to have largely unexplained infertility.Approximately 10 percent of the female population is believed to sufferfrom premature aging ovaries and this diagnosis is often mistaken forunexplained infertility (Nikolaou and Templeton, 2003, Gleicher N.,2005). Patient B later developed signs of prematurely aging ovaries and,finally, showed elevated FSH levels. In the time sequence in which allof these observations were made, Patient B followed the classicalparallel premature aging curve (Nikolaou and Templeton, 2003; GleicherN., 2005).

Once substituted with oral DHEA a reversal of many findingscharacteristic of the aging ovary was noted. DHEA treatment includesadministering a dose of between about 50 mg/day and about 100 mg/day,preferably between about 60 mg/day and about 80 mg/day, and in one studyabout 75 mg/day to a human female. The DHEA dose could be administeredas a single dose or as multiple doses over the course of a day.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a significant effect afterabout 8 weeks or about 2 months of administration, but its effect maycontinue to increase to about four months, and further may continue pastfour months of use. Specifically, DHEA treatment has a statisticallysignificant effect after about at least 16 weeks or about at least 4months of administration.

After DHEA administration, Patient B's DHEA and dehydroepiandrosteronesulfate (DHEAS) levels normalized. In subsequent natural cycles anapparently normal spontaneous follicular response was observed, withnormal ovulatory estradiol levels in a patient with persistently lowestradiol levels before DHEA treatment.

DHEA deficiency may be a cause of female infertility and may be apossible causative agent in the aging processes of the ovary. The casestudy involving Patient B also presents further confirmation of thevalue of DHEA substitution whenever the suspicion exists that ovariesmay be lacking of DHEA substrate. Since the process is familial(Nikolaou and Templeton, 2003), it is reasonable to assume that, likeother adrenal enzymatic defects, 17,20-desmolase deficiency may occureither in a sporadic or in an inherited form. As both forms will resultin abnormally low DHEA levels, both may lead to phenotypical expressionas premature ovarian aging.

VII. Increase in Spontaneous Conceptions

Additionally, with DHEA treatment, there may be an unexpectedly largenumber of spontaneous conceptions in women waiting to go into an IVFcycle. DHEA treatment includes administering a dose of between about 50mg/day and about 100 mg/day, preferably between about 60 mg/day andabout 80 mg/day, and in one study about 75 mg/day to a human female.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a more significant effectafter about 8 weeks or about 2 months of administration, but its effectmay continue to increase to about four months, and further may continuepast four months of use. Specifically, DHEA treatment has astatistically significant effect after about at least 16 weeks or aboutat least 4 months of administration.

The DHEA treatment may be at least about 2 weeks before spontaneousconception occurs. In the population of women who are waiting to go intoIVF, the spontaneous pregnancy rate is a fraction of about 1% per month.However, in the population of women who have been on DHEA treatment,there were 13 spontaneous pregnancies out of 60 women. As such, DHEAtreatment increases spontaneous pregnancies in one study at least about21 fold. This provides evidence that DHEA works not only in conjunctionwith gonadotropin stimulation of ovaries, but also without gonadotropinstimulation of ovaries.

VIII. Increase in Male Fetus Sex Ratio

A further effect of DHEA treatment is raising androgen levels in afemale to increase the male fetus sex ratio. The gender of offspring maynot be solely determined by chance. More highly androgenized femalemammals give birth to more male offspring. Androgens, such as DHEA, maybe utilized and an elevated baseline level of above about 250 ng/dl,preferably above about 350 ng/dl, may be sufficient. Infertile womenwith diminished ovarian reserve established a human model to investigatethis theory. Data obtained from this model support an effect ofandrogenization on gender not through a follicular selection mechanismbut rather through different mechanisms than previously theorized asevidenced by occurring after the preimplantation embryo stage.

Routine treatment protocol involves administering about 25 mg ofmicronized, pharmaceutical grade DHEA, TID, to a human female touniformly raise levels of unconjugated DHEA above 350 ng/dl and,therefore, raise baseline testosterone. In six pregnancies spontaneouslyconceived, the distribution between female and male offspring was equal,at three and three, respectively. In contrast, in the remaining 15offspring, which were products of pregnancies achieved through IVF, thedistribution was 12 males and 3 females (p=0.035). Amongst womenundergoing IVF and PGD, 53 embryos were analyzed from 17 IVF cycles, allhaving undergone ICSI. The gender distribution was not significantlyskewed, with 27 being male and 26 female.

The data, demonstrating a strong trend towards both significance overalland significance (p=0.035) amongst IVF patients, suggest that genderdetermination may be influenced through hormone environments. The evendistribution of gender (27 male and 26 female) in this group of patientsargues against a selection process towards male, which is driven by thefollicular environment, as has been previously suggested. The evendistribution of gender in preimplantation embryos, seen in the controlgroup, also speaks against such an effect.

The only remaining conclusion from the here presented data is thatfemale androgenization affects gender selection after thepreimplantation embryo stage and that, by definition, identifies thestage of androgenic influence on gender at or after implantation. Allbut one IVF cycles in study and control groups underwent IC SI, whichrequires the removal of granulose cells from the oocyte. One hypothesisis that such a removal may render the local environment more favorabletowards the implantation of male than female embryos. A secondhypothesis would suggest a similar effect, based on the difference inhormonal milieu in the luteal phase between IVF and spontaneousconception cycles, with the former uniformly supported by progesteroneand the latter only sporadically, or not at all. The data providesevidence that the androgenization of females may increase the prevalenceof male offspring, especially with IVF.

IX. Increase in Pregnancy Rates

An additional benefit of DHEA treatment is an unexpectedly high numberof pregnancies in women, particularly in women with diminished ovarianfunction. DHEA supplementation is also associated with increasedcumulative pregnancy rates and a shorter interval to pregnancy amongwomen with evidence of decreased ovarian function entering evaluationand treatment for infertility.

DHEA treatment includes administering a dose of between about 50 mg/dayand about 100 mg/day, preferably between about 60 mg/day and about 80mg/day, and in one study about 75 mg/day to a human female. Further,DHEA may be administered in a time-release formulation, over the courseof the day, or in a single dose. For example, the about 75 mg/day couldbe administered in a single dose of 75 mg or could be administered as 25mg three times throughout the day. Particularly, the DHEA treatment maybe administered to a premenopausal woman with diminished ovarianfunction. DHEA may have an effect after about 4 consecutive weeks.However, DHEA has a significant effect after about 8 weeks or about 2months of administration, but its effect may continue to increase toabout four months, and further may continue past four months of use.Specifically, DHEA treatment has a statistically significant effectafter about at least 16 weeks or about at least 4 months ofadministration.

A case control study of 190 women over 30 years old with diminishedovarian function were studied between 1999 and December 2005. The studygroup included 89 patients with a mean age of about 41.6 who usedsupplementation of about 75 mg daily of oral, micronized DHEA for up tofour months prior to entry into IVF. The control group composed 101patients with a mean age of about 40.0 who received infertilitytreatment but did not use DHEA. The primary outcome was clinicalpregnancy after the patient's initial visit.

Ovarian stimulation was identical for study and control groups andconsisted of microdose agonist flare, followed by maximal dosagegonadotropin stimulation, using about 300-450 IU of FSH and about 150 IUof HMG. Study patients received DHEA continuously until a positivepregnancy test was obtained or until the patient dropped out oftreatment.

Using a developed Cox proportional hazards model, the proportionalhazards of pregnancy among women using DHEA was compared with thecontrols group. The results were that cumulative clinical pregnancyrates were significantly higher in the study group (25 pregnancies of 89patients for 28% vs. 11 pregnancies of 101 patients for 11%; relativehazard of pregnancy in study group (HR 3.8; 95% CI 1.2 to 11.8;p<0.05)). Specifically, about 28% of the patients that received DHEAachieved a clinical pregnancy, and about 11% of the patients that didnot receive DHEA achieved clinical pregnancy. As such, DHEA treatmentincreases the percentage of clinical pregnancies between about 130% andabout 180%, preferably between about 140% and about 170%, and in onestudy about 157%. As such, DHEA treatment increases clinical pregnanciesby at least about 150%.

Further, the results of this study show a statistically significantpercentage of women that achieved clinical pregnancy only with DHEAtreatment. See Table 8 in Example 7 herein. Table 8 shows 25 of 89 womenin the DHEA treated group achieving clinical pregnancy, including 6 of16 with no other treatment other than DHEA, and 6 of 9 women hadintrauterine insemination (IUI/COH) but no IVF. About at least one-halfof the patients (or at least about 50% of the patients), a total of 12out of the 25 women (about 6 of 16 women with no other treatment, andabout 6 of 9 women treated with intrauterine insemination) thatestablished pregnancy did so spontaneously (i.e., with no IVFtreatment). As such, DHEA treatment also increases the percentage ofclinical pregnancies and significantly reduces the cumulative time topregnancy.

Along with increased clinical pregnancies, women in this study, with amean age of about 41.6, which were treated with DHEA had decreasedmiscarriage rates. Specifically, approximately 36% of the women in thecontrol group (4 of 11 women) that did not receive DHEA had miscarriagesand, in comparison, only approximately 20% of the women in theDHEA-treated group (5 of 25 women) had miscarriages. As such, DHEAtreatment decreased the miscarriage rate between about 30% and about60%, preferably between about 40% and about 50%, and in one study about44%. DHEA treatment decreases the miscarriage rate by at least about ⅓,and preferably by at least about ½.

The data, described further herein, provides evidence that the DHEAsupplementation improves spontaneous pregnancy rates, IVF pregnancyrates, cumulative pregnancy rates, and decreases the time interval topregnancy.

X. Decrease in Miscarriage Rates

Supplementation with dehydroepiandrosterone (DHEA) as described hereinbelow decreases miscarriage rates in infertile women with diminishedovarian reserve. DHEA administration, for an average of at least 2months, decreases the miscarriage rate. DHEA treatment includesadministering a dose of between about 50 mg/day and about 100 mg/day,preferably between about 60 mg/day and about 80 mg/day, and in one studyabout 75 mg/day to a human female. Further, DHEA may be administered ina time-release formulation, over the course of the day, or in a singledose. For example, the about 75 mg/day could be administered in a singledose of 75 mg or could be administered as 25 mg three times throughoutthe day. Particularly, the DHEA treatment may be administered to apremenopausal woman with diminished ovarian function. DHEA may have aneffect after about 4 consecutive weeks. However, DHEA has a moresignificant effect after about 8 weeks or about 2 months ofadministration, but its effect may continue to increase to about fourmonths, and further may continue past four months of use. Specifically,DHEA treatment has a statistically significant effect after at leastabout 16 weeks or at least about 4 months of administration, andpreferably, DHEA treatment is administered for at least about 16consecutive weeks or at least about 4 months.

About 85% of miscarriages are due to chromosomal abnormalities. As such,decreasing the miscarriage rates in women may indicate a decrease inaneuploidy rates.

After about at least two months of prior DHEA supplementation, the rateof clinical miscarriages in 73 pregnancies, established at twoindependent fertility centers in the United States (U.S.) and Canada,was compared to the national U.S. miscarriage rates, reported for invitro fertilization (IVF) pregnancies for the year 2004.

The reduction in miscarriage rates in DHEA pregnancies at both centerswere similar (15.0% and 15.2%) for a combined reduction in miscarriagerates of about 15.1%. The Mantel-Haenszel common odds ratio (and 95% CI)for the odds of miscarriage with DHEA supplementation, stratified byage, was significantly lower relative to the odds of miscarriage in thegeneral U.S. IVF population [0.49 (0.25-0.94; p=0.04)]. Miscarriagerates after DHEA supplementation was lower at all ages than the 2004 USnational averages, but the difference was more pronounced above age 35years.

More specifically, DHEA treatment decreases the miscarriage rate forwomen under the age of about 35 between about 5% and about 25%,preferably between about 10% and about 20%, and in one study about15.7%. DHEA treatment decreases the miscarriage rate for women under theage of about 35 by at least about one-seventh. Further, DHEA treatmentdecreases the miscarriage rate for women between the ages of about 35and about 37 between about 50% and about 70%, preferably between about55% and about 65%, and in one study about 60.8%. DHEA treatmentdecreases the miscarriage rate for women between the ages of about 35and about 37 by at least about one-half. Also, DHEA treatment decreasesthe miscarriage rate for women between the ages of about 38 and about 40between about 20% and about 40%, preferably between about 25% and about35%, and in one study about 31.6%. DHEA treatment decreases themiscarriage rate for women between the ages of about 38 and about 40 byat least about ¼, and preferably by at least about ⅓. Additionally, DHEAtreatment decreases the miscarriage rate for women between the ages ofabout 41 and about 42 between about 30% and about 60%, preferablybetween about 40% and about 50%, and in one study about 45.3%. DHEAtreatment decreases the miscarriage rate for women between the ages ofabout 41 and about 42 by at least about ⅓, and preferably by at leastabout ½. Further, DHEA treatment decreases the miscarriage rate forwomen over the age of about 42 between about 40% and about 60%,preferably between about 45% and about 55%, and in one study about50.1%. DHEA treatment decreases the miscarriage rate for women over theage of about 42 by at least about ½.

DHEA supplementation is associated with a significantly decreasedmiscarriage rate in women, especially above the age of about 35. DHEAtreatment decreases the miscarriage rate for women over the age of about35 by at least about 30% or at least about ⅓. Supplementation with DHEAreduces the miscarriage risk in this high risk population to levelsreported for the general population.

This observation supports a beneficial effect of DHEA on aneuploidyrates. DHEA treated women with diminished ovarian reserve, who producefew embryos, only rarely qualify for preimplantation genetic screening.Data accumulation on embryo aneuploidy rates is, therefore, difficult.Because embryo aneuploidy rates are reflected in miscarriage rates, bydemonstrating a remarkable reduction in miscarriage rates, there iscircumstantial evidence that DHEA supplementation may reduce the rate ofaneuploid embryos in infertile women.

XI. More on Decreasing Miscarriage Rates

Dehydroepinadrosterone (DHEA) supplementation improves pregnancy chancesin women with diminished ovarian reserve (DOR) by possibly reducinganeuploidy. Since a large majority of spontaneous miscarriages areassociated with aneuploidy, one can speculate that DHEA supplementationmay also reduce miscarriage rates.

We retroactively compared, utilizing two independent statistical models,miscarriage rates in 73 DHEA supplemented pregnancies at two independentNorth American infertility centers, age-stratified, to miscarriagesreported in a national U.S. in vitro fertilization (IVF) data base.

After DHEA supplementation the miscarriage rate at both centers was15.1% (15.0% and 15.2%, respectively). For DHEA supplementationMantel-Hanszel common odds ratio (and 95% confidence interval),stratified by age, was significantly lower, relative to odds ofmiscarriage in the general IVF control population [0.49 (0.25-0.94;p=0.04)]. Miscarriage rates after DHEA were significantly lower at allages but most pronounced above age 35 years.

Since DOR patients in the literature are reported to experiencesignificantly higher miscarriage rates than average IVF patients, thehere observed reduction in miscarriages after DHEA supplementationexceeds, however, all expectations. Miscarriage rates after DHEA notonly were lower than in an average national IVF population but werecomparable to rates reported in normally fertile populations. Lowmiscarriage rates, comparable to those of normal fertile women, arestatistically impossible to achieve in DOR patients without assumptionof a DHEA effect on embryo ploidy. Beyond further investigations ininfertile populations, these data, therefore, also suggest theinvestigations of pre-conception DHEA supplementation in normal fertilepopulations above age 35 years.

XII. Improvement in Ovarian Reserve

Our study presents the first objective evidence that supplementationwith dehydroepiandrosterone (DHEA) of women with diminished ovarianreserve (DOR) improves ovarian reserve at all ages.

Our objective was to determine whether supplementation withdehydroepiandrosterone (DHEA) of women, suffering from diminishedovarian reserve (DOR), objectively improves ovarian reserve, based onanti-Müllerian hormone levels (AMH).

120 consecutive women, presenting with DOR were patients in this study.We administered DHEA to each patient to improve ovarian reserve.

DHEA administration, for an average of at least about 1 month, improvesovarian reserve. Preferably, DHEA administration lasts for between about15 days to about 150 days, more preferably between about 25 days and 130days, and in one study between about 30 days and about 120 days (mean 73days±27 days).

DHEA administration also includes administering a dose of between about50 mg/day and about 100 mg/day, preferably between about 60 mg/day andabout 80 mg/day, and in one study about 75 mg/day to a human female.Further, DHEA may be administered in a time-release formulation, overthe course of the day, or in a single dose. For example, the about 75mg/day could be administered in a single dose of about 75 mg or could beadministered as about 25 mg three times throughout the day.Particularly, the DHEA treatment may be administered to a premenopausalwoman with diminished ovarian function. DHEA may have an effect afterabout 4 consecutive weeks. However, DHEA has a more significant effectafter about 8 weeks or about 2 months of administration, but its effectmay continue to increase to about four months, and further may continuepast four months of use. Specifically, DHEA treatment has astatistically significant effect after at least about 16 weeks or atleast about 4 months of administration, and preferably, DHEA treatmentis administered for at least about 16 consecutive weeks or at leastabout 4 months.

Our main outcome measure was AMH levels in relationship to DHEAsupplementation over days of DHEA supplementation using linearregression and, in longitudinal evaluation, by examining the interactionbetween days of DHEA treatment and pregnancy success in respect tochanges in AMH levels.

Our results were that AMH levels significantly improved after DHEAsupplementation over time (p=0.002). Age (p=0.007) and length oftreatment (p=0.019) were independently associated with increasing AMH.Women under about age 38 years demonstrated higher AMH levels andimproved AMH proportionally more than older females. Longitudinally, AMHlevels improved by approximately 60 percent from 0.22±0.22 ng/ml to0.35±0.03 ng/ml (p<0.0002). Women who reached IVF experienced a 23.64%clinical pregnancy rate. Those who conceived improved AMH significantlymore than women who did not (p=0.001).

In sum, DHEA supplementation significantly improves ovarian reserve withDOR. Additionally, improvement increases with longer DHEAsupplementation and is more pronounced in younger women under age about38 years.

The following examples are to be construed as merely illustrative andnot limitative of the disclosure in any way.

Example 1 Improved Ovulation

A study including a 43 year old woman, Patient A, undergoing IVF withbanking of multiple cryopreserved embryos for future aneuploidy screenand transfer is administered an androgen, namely DHEA. In ten months sheundergoes eight treatment stimulation cycles while continuouslyimproving her ovarian response, resulting in oocyte and embryo yieldsfar beyond those previously seen in a woman her age.

Patient A's history is unremarkable except for two previous malarialinfections. She is allergic to sulfa medications and has a history ofenvironmental allergies. Her surgical history includes umbilical herniarepair at age one and cholecystectomy at age 21. She had used oralcontraceptives for over 10 years. She has no history of irregularmenstrual cycles.

Day three serum FSH and estradiol (E2) in her first IVF cycle are 11mIU/ml and 18 pg/ml, respectively. In subsequent cycles her baseline FSHis as high as 15 mIU/ml. She is given an ovulation induction protocolwhich is prescribed for patients with evidence of decreased ovarianreserve. Briefly, the protocol includes the following medications:norethindrone acetate tablets (10 mg) for 10 days, starting on day twoof menses, followed three days later by a “microdose” dosage of 40 μg ofleuprolide acetate, twice daily, and, after another three days, by 600IU of FSH (Gonal-F; Ares-Serono, Geneva, Switzerland) daily. Peak serumE2 concentration on day nine of stimulation is 330 pg/ml. Followinginjection of 10,000 IU human chorionic gonadotropin (hCG), she undergoesoocyte retrieval. Only one oocyte is obtained and one embryo iscryopreserved.

Because of the poor response to ovulation stimulation, she is advised toconsider donor oocyte or embryo donation. She rejects both options. Shestarts a second cycle using the same stimulation protocol with oneexception: instead of 600 IU of FHS daily, Patient A received 450 IU ofFSH and 150 IU of human menopausal gonadotropin (HMG, Pergonal,Ares-Serono, Geneva, Switzerland). This stimulation protocol iscontinued in identical fashion for the remaining cycles. However, twoweeks before starting her second cycle, she begins administration of 75mg per day of oral micronized DHEA. The date on which she beginsadministration of 75 mg per day of oral micronized DHEA is Oct. 6, 2003.

Methods of Example 1

The eight treatment cycles are divided into three groups to allowstatistical comparison: pre-initiation and very early use of DHEA(early=cycles 1 and 2), initial cycles (mid=cycles 3-5), and latercycles (late=cycles 6-8). Comparison between these categories is byone-way analysis of variance (ANOVA) and multiple comparisons byStudent-Neuman-Keuls (SNK) test. The homogeneity of variances and usedorthogonal linear contrasts are tested to compare groups and polynomialcontrast to test for linear and quadratic trends. All outcomes arepresented as mean±1 standard deviation. Rate of change of oocyte counts,cryopreserved embryos and (log transformed) peak estradiol betweensubsequent cycles is estimated by linear regression.

Embryos are evaluated by the embryologists on day threepost-insemination for cell-count and grading. Embryo grading is based ona 1 to 4 scale depending on symmetry, percent fragmentation andappearance of the cytoplasm. All viable embryos are cryopreserved.Statistics are performed using SPSS for Windows, Standard version 10.0.7(SPSS Co., Chicago, Ill.). Assay of E2 and FSH are performed using theACS: 180 chemoluminescence system (Bayer Health Care LLC, Tarrytown,N.Y.).

A method of preconditioning ovulation induction in a human female isconceived, comprising administering an androgen in a female for at leastabout four consecutive months. In one embodiment, the androgen is DHEA.Administration of DHEA for at least about four consecutive months mayfurther comprise administering high dose gonadotropins to the female.Furthermore, DHEA may be administered along with follicle stimulatinghormone, human menopausal gonadotropin, norethindrone acetate,leuprolide acetate, and human chorionic gonadotropin. DHEA may beadministered orally.

The length of time the androgen is administered to the female can be atleast four consecutive months. The DHEA treatment may continue for morethan four months. In one embodiment, the androgen administered is DHEA.

Results of Example 1

The results of ovulation induction are displayed in FIG. 1. After eightstimulation cycles and approximately eight months of DHEA treatment,Patient A produced 19 oocytes and 11 cryopreservable embryos. A total of50 viable embryos have so far been cryopreserved. Significantly moreoocytes (p=0.001) and cryopreserved embryos (p<0.001) are obtained inthe late cycles (cycles 6-8, 4+ consecutive months of DHEA treatment)compared to the combined early and mid cycles (cycles 1-5, 0-4consecutive months of DHEA treatment). There is no significantdifference in average embryo cell count (6.83±1.37 vs. 7.2±1.15) ormorphology (3.6±0.5 vs. 3.7±0.5) between early and mid compared to latecycles. Peak E2, total oocyte, and embryos cryopreserved increaselinearly from cycle to cycle, as shown in FIG. 1. Oocyte yield increase2.5±0.34 oocytes per cycle (p<0.001), cryopreservable embryo yieldincrease 1.4±0.14 embryos per cycle (p<0.001) and (log) peak E2 increase0.47±0.06 (p<0.001) across treatment cycles.

The linear increase in (log) peak E2 shown in FIG. 2 represents a cycleto cycle rate of increase from 123 pg/ml/cycle to 1491 pg/ml/cycle overthe eight cycles of treatment. After adjusting for cycle day, the(harmonic) mean E2 is 267 pg/ml (95% confidence intervals (CI) 143 to498 pg/ml) in the early phase, 941 pg/ml (95% CI 518 to 1712 pg/ml) inthe mid phase, and 1780 pg/ml (95% CI 1121 to 2827 pg/ml) in the latephase of treatment. Each of these homogeneous subsets is significantlydifferent from the other (p<0.05) by SNK multiple comparison testing.

The dramatic increase in oocyte and embryo yield experienced by this 43year old woman is completely surprising and unexpected. Patient A'spost-DHEA response to ovulation induction has become more like that of ayounger woman with PCO, than that of a 43 year old woman. Since startingDHEA treatment, Patient A has produced 49 embryos of high enough qualityto undergo cryopreservation. Sixty percent of those embryos wereproduced in the last three cycles of treatment, which took place afterat least about four consecutive months after starting treatment. Afterproducing only one embryo prior to starting DHEA treatment, Patient Aimproved by an order of magnitude and produced 13 oocytes and 9 embryosin a cycle after at least about four consecutive months of DHEAtreatment, 16 oocytes and 10 embryos in a cycle after at least aboutfive and a half consecutive months of DHEA treatment, and 19 oocytes and11 embryos in a cycle after at least about seven consecutive months ofDHEA treatment.

The increasing numbers of cryopreservable embryos due to DHEAsupplementation suggest improved embryo quality.

Example 2 Improved Oocyte Fertilization and Cumulative Embryo Score

In another study, thirty (30) patients with evidence of decreasedovarian reserve were given supplemental DHEA 25 mg three times a day,for a total of 75 mg per day, for an average of about 4 months beforebeginning ovulation induction for IVF. Twelve patients contributed datafrom cycles both pre-DHEA and post-DHEA, eleven patients contributeddata from cycles only pre-DHEA, and seven patients contributed data fromcycles only post-DHEA. Patients' response to ovulation induction beforeDHEA treatment was compared to patients' response to ovulation inductionafter DHEA treatment with regard to peak estradiol, oocyte production,and embryos transferred and embryo quality.

The thirty patients contributed to data for 42 total cycles, 23 cyclesprior to and 19 cycles after starting DHEA supplementation. In comparingthe patients as a group pre- and post-DHEA treatment cycles, there wereimprovements in cancellation rate, peak estradiol, average day 3 embryocell counts, and embryo grade. However, average oocyte numbers, eggsfertilized, day-three embryos, embryos transferred and cumulative embryoscores increased significantly after DHEA treatment. In logisticregression models adjusted for oocyte number, there was evidence ofimproved fertilization rates (p<0.005), increased numbers of day-threeembryos (p<0.05), and of improved overall embryo score (p<0.01). In 34IVF cycles that reached the embryo transfer stage, a positive pregnancytest was obtained in zero of 16 cycles with less than an average ofabout 4 months of DHEA treatment and in 4/18 cycles after an average of4 months of DHEA treatment.

This case series illustrates that some ovarian function can be salvaged,even in women of advanced reproductive age.

TABLE 1 Univariate comparison of results of in vitro fertilizationbefore and after treatment with DHEA. Pre DHEA Post DHEA p N 23 19 Age40.9 ± 0.7  42.8 ± 0.7  ns Weeks of DHEA — 16.1 ± 2.4  — Cancellation5/21 (21%) 1/19 (5%) ns Peak Estradiol 1018 ± 160  1192 ± 904  nsOocytes 3.3 ± 0.7 5.8 ± 1.0 0.04 Fertilized eggs 1.3 ± 0.3 4.6 ± 0.8<0.001 Average Day 3 3.1 ± 0.6 4.5 ± 0.5 ns embryo cell count AverageDay 3 2.4 ± 0.3 2.8 ± 0.3 ns embryo grade Cumulative  34 ± 6.8   98 ±17.5 0.001 Embryo Score Transferred embryos 1.0 ± 0.2 2.6 ± 0.4 0.001Number of Day 3 0.9 ± 0.2 3.2 ± 0.6 0.001 Embryos Positive hCG 0/16 4/18ns (per transfer cycle)

Cycle characteristics and responses to treatment are shown in Table 1.The average age of the patients who began DHEA was 41.6±0.6 years. Womenin the DHEA group used DHEA for a median value of 16 weeks before theirIVF cycle. The cycle cancellation rate was 5 of 21 cycles (21%) pre-DHEAand 1 of 19 (5%) post-DHEA. There was no statistically significantdifference in peak estradiol levels between pre- and post-DHEA cycles.

Continuing with the cycle outcomes presented in Table 1, there areimprovements in average cell count of day-three embryos and mean embryograde after DHEA treatment, however the differences are not significant.Mean oocyte numbers, fertilized eggs, day-three embryos, embryostransferred and cumulative embryo scores, all increased significantlyafter DHEA treatment. In the models adjusted for oocyte number, therewas still evidence of increased fertilization rates (1.93 fertilizedoocytes, 95% C.I. 0.82-3.04; p<0.005), increased numbers of day-threeembryos (1.36 embryos, 95% C.I. 0.34-2.4; p<0.05), and of improvedoverall embryo score (32.8, 95% C.I. 9.6-56; p<0.01).

FIG. 3 shows paired comparisons of fertilized oocytes (average increase2.5±0.60; p=0.002) among 12 patients with DHEA treatment cycles of lessthan about 4 weeks to fertilized oocytes in the same 12 patients afterat least about 4 weeks of DHEA treatment. FIG. 4 shows pairedcomparisons of day 3 embryos (average increase 2.0±0.57; p=0.005) among12 patients with DHEA treatment cycles of less than about 4 weeks and atleast about 4 weeks during IVF cycles. The paired comparisons shows thatthe mean increase in the number of fertilized oocytes was modest, butsignificant, (1.42±0.63 increased numbers of fertilized oocytes;p<0.05).

The mean increase in embryo scores was 57±14.7 (p<0.01). The increase inthe number of day 3 embryos was 2.0±0.57 (p=0.005) (See FIG. 4) and theincreased fertilization quantity was 2.5±0.60 fertilized oocytes perpatient (p=0.002) (See FIG. 3). DHEA supplementation improves theaverage oocyte numbers, eggs fertilized, day three embryos, embryostransferred, and cumulative embryo score.

In addition, DHEA supplementation also improves pregnancy rates anddecreases time to pregnancy. Two patients achieved ongoing pregnancieswhile taking DHEA without IVF; one (43 year old) while using DHEA duringa stimulated IUI (intrauterine insemination) cycle and a second (37 yearold) conceived spontaneously following an unsuccessful IVF cycle. Athird patient (40 year old) also conceived spontaneously while preparingfor an IVF cycle; however that pregnancy ended in a spontaneousabortion. In all 7 of 45 (16%) patients using DHEA have conceived and 5of 45 patients (11%) have experienced continuing pregnancies.

Example 3 Increased Euploidy Rate

In another study (data not shown), patients were analyzed after fourweeks of DHEA treatment. Seven patients had embryos tested bypre-implantation genetic diagnosis (PGD). In three women who had PGDafter less than four weeks of DHEA usage and a mean age 41.5±5.1 at thetime of starting IVF cycles, the euploidy, or normal chromosome number,rate was 2/30 embryos (6.6%). In six patients who had PGD after morethan four weeks of DHEA usage, and a mean age of 43.7±1.3 years at thetime of starting IVF cycles, the euploidy rate increased to ( 8/27;29.6%), though this trend did not reach statistical significance. Thereis a mean age difference between patients who underwent IVF after lessthan four weeks of DHEA usage (mean age 41.5±5.1) and patients whounderwent IVF after at least four weeks of DHEA usage (mean age43.7±1.3).

As women age, there is a substantial decline in euploidy rates inembryos produced. Thus, the increase in euploidy results in older womenis dramatic evidence of the effectiveness of DHEA in improving embryoquality.

Example 4 DHEA Treatment Increases Euploidy Number

In a series of studies, it has been documented that DHEA supplementationin women with diminished ovarian reserve (DOR) increases egg and embryocount, improves egg and embryo quality, increases pregnancy rates, andshortens time to conception.

The reports of the studies point towards improvements in follicularrecruitment after treatment with androgenic compounds. Since DHEAeffects are statistically significant after approximately four months,and since this time period is approximately reflective of the fullfollicular recruitment cycle, we concluded that DHEA may, at least inpart, affect follicular recruitment processes, possibly by influencingapoptosis. Androgens have been reported to affect granulosa cellapoptosis.

While women with prematurely DOR appear to have normal embryonicaneuploidy rates, older women, with physiologic aging ovaries,demonstrate very high aneuploidy rates of their embryos. Increasinganeuploidy rates with advancing female age are, therefore, considered aprimary cause for diminishing pregnancy chances, and an increasingmiscarriage risk, in older women. Since treatment with androgeniccompounds in such patients appears to improve embryo quality andpregnancy chances, it is likely that such treatment positively affectsaneuploidy rates.

Materials and Methods of Example 4

All the IVF cycles performed at the Center for Human Reproduction (CHR)in New York, N.Y., between 2004 and 2006 for cycles performed in womenwith a diagnosis of DOR were retroactively reviewed. The studypopulation, involving 27 IVF cycles, was selected amongst those cycleswhich, in addition, had undergone preimplantation genetic diagnosis(PGD).

The diagnosis of DOR was made based on previously reported abnormallyhigh, age stratified baseline FSH levels. In practical terms, this meantthat a diagnosis of DOR was reached if baseline FSH levels exceeded the95% confidence interval of age appropriate levels, independent of priorIVF retrievals and/or oocyte numbers. At, or above age 43, all patientswere considered to suffer from DOR, independent of baseline FSH level.

Since the year 2004, women with proven DOR, who had undergone at leastone prior ovarian stimulation, demonstrating ovarian resistance based oninadequately low oocyte numbers, routinely were offered oral DHEAsupplementation (25 mg TID) prior to any further IVF cycle starts. Ifunder age 40, DHEA was given for up to four months prior to IVF. Womenof older age received DHEA, if possible, for at least two months.

Women with DOR, who had no proof of ovarian resistance, were not placedon DHEA supplementation until such proof was obtained, unless they wereat, or above, age 43 years. IVF cycles on DHEA supplementation have,therefore, to be considered as more severely affected by DOR than thosecycles that were conducted without such supplementation. This fact isalso reflected by the baseline cycle characteristics of DHEA-treated,and -untreated, patients (Table 2), which demonstrate trends towardsolder age and higher baseline FSH levels in DHEA treated patients.

TABLE 2 Baseline characteristics of DHEA-treated, and -untreated,patients¹ DHEA-TREATED DHEA-UNTREATED n = 8 n = 19 Age (± SD, year) 41.2± 4.7 38.9 ± 5.1  Baseline FSH² ± SD 12.4 ± 9.2 9.0 ± 2.7 (mIU/ml)Baseline Estradiol² ± SD  59.7 ± 32.2 68.1 ± 59.1 (pg/ml) ¹None of thebaseline parameters, listed in the table, differed to a statisticallysignificant degree between the two groups. ²Reflects highest baselinelevel of each patient, and not necessarily the baseline level of the IVFcycle.

For the purpose of this analysis, a patient had to be for at least onemonth (30 days) on DHEA supplementation in order for the IVF cycle to beconsidered amongst DHEA-treated cycles. All other DOR patients wereconsidered to have received no DHEA treatment. Following thisdefinition, 19 DOR patients had received no DHEA supplementation, andeight had.

All women with DOR, independent of DHEA supplementation, were stimulatedwith identical protocols, as previously reported in detail elsewhere. Inshort, they, without exception, received a microdose agonist protocolwith maximal gonadotropin stimulation of 600 IU to 750 IU daily, withpreponderance of FSH, and a smaller daily amount of human menopausalgonadotropin (hMG).

PGD was performed in routine fashion, as also previously described indetail, and involved the analysis of chromosomes X, Y, 13, 16, 18, 21and 22 by fluorescence in situ hybridization (FISH) on day three afterfertilization. Embryo transfer occurred on day five after fertilization.

Patients were represented by only one cycle outcome in each group. Ifpatients had undergone more than one cycle, either with, or without,DHEA supplementation, only their latest cycle was included in theanalysis. Three patients underwent both a pre-DHEA and a post-DHEA cycleand in those cases both cycles were included in the analysis.

Statistical analysis was performed using SPSS for windows, standardversion 10.0.7. Data are presented as mean±one standard deviation,unless otherwise noted, and statistical differences between the twostudy groups were tested by Chi-square and (two-sided) Fisher's ExactTest, where applicable, with significance being defined as p<0.05.

Results of Example 4

A total of 27 consecutive IVF cycles in women with DOR who also hadundergone preimplantation genetic diagnosis (PGD) were identified andevaluated. Amongst those, 19 had entered IVF without DHEA treatment and8 had received DHEA supplementation for at least four weeks prior to IVFstart.

Table 3 summarizes cycle outcomes.

TABLE 3 IVF cycle and PGD outcomes DHEA-TREATED DHEA-UNTREATED PeakEstradiol ± SD 2310.3 ± 1108.1 2123.3 ± 1054.7 (pg/ml) Oocytes ± SD 10.4± 7.3  8.5 ± 4.6 Embryos ± SD¹ 9.1 ± 7.3 5.7 ± 2.7 n Euploid ± SD 2.1 ±1.4 1.6 ± 2.3 % Euploid ± SD 44.1 ± 37.8 21.4 ± 27.5 n Aneuploid ± SD4.4 ± 3.0 3.5 ± 0.3 % Aneuploid ± SD 55.9 ± 37.8 78.6 ± 27.5 Patientswith euploid 8/8 (100)² 7/13 (53.8)² embryos (%) SD, standard deviationof mean; ¹Reflects total number of embryos. Since only high quality6-cell to 8-cell day-3 embryos undergo PGD, the number of embryos testedfor ploidy was smaller. ²Reflects a statistically significant differenceby Likelihood ratio (p = 0.004) and (two-sided) Fisher's Exact Test; p =0.026. Other comparisons in this table did not reach statisticalsignificance.

DHEA treatment resulted in trends towards higher oocyte numbers(10.4±7.3 vs. 8.5±4.6). A significantly larger number of DHEA treatedIVF cycles (eight out of eight, 100%) had at least one euploid embryofor transfer than in untreated cycles (10/19, 52.6%; Likelihood ratio,p=0.004; Fisher's Exact Test, p=0.026). In other words, the primaryresult reaching statistical significance was the difference in thepercentage of IVF cycles which resulted in the transfer of at least oneeuploid embryo, with DHEA treated patients reaching embryo transfer in100 percent of cycles, while untreated patients did so in only 52.6percent of cases.

As can be seen in Table 3, peak estradiol levels, oocyte and embryonumbers and the results of PGD, all demonstrated trends towards abeneficial effect of DHEA. Peak estradiol levels were higher and oocyte,as well as embryo numbers, were larger. There was also a trend towardsmore euploidy in embryos from treated cycles, both in absolute numbersand in percentages of embryos evaluated by PGD.

Amongst the 27 reported cycles, three patients contributed pre- andpost-DHEA cycles. When these cycles were separately analyzed, theydemonstrated similar trends as observed for the whole study (Table 4).

TABLE 4 IVF cycle parameters in 3 women with DHEA and -no-DHEA cycles¹Age ± SD (years) 38.2 ± 5.5 Baseline FSH² ± SD (mIU/ml) 10.5 ± 1.5Baseline Estradiol² ± SD (pg/ml)  54.4 ± 21.7 DHEA-TREATEDDHEA-UNTREATED Time pre-/post DHEA 2.4 ± 2.5 1.9 ± 2.2 (months) Oocytes± SD 6.0 ± 4.8 4.8 ± 1.0 Total Embryos ± SD 4.0 ± 2.7 4.5 ± 0.6Aneuploid Embryos 2.0 ± 1.8 3.5 ± 0.6 SD, standard deviation; ¹None ofthe differences between the two study groups reached statisticalsignificance, ²Reflects highest baseline level of patients, but notnecessarily baseline level during IVF cycle.

Discussion of Example 4

The here presented study demonstrates evidence that DHEA improves, to astatistically significant degree, the number of euploid embryosavailable for embryo transfer after IVF, and may be at least a partialexplanation of why DHEA supplementation improves pregnancy chances inwomen with DOR. The study also demonstrates a trend towards higherpercentages of euploid embryos after DHEA and higher absolute numbers ofeuploid embryos. The here observed effect of statistically moretransferable, euploid embryos, may be due to larger oocyte and embryonumbers, lower aneuploidy rates, or both effects combined.

The mean number of euploid embryos increased after DHEA treatment byapproximately one-half embryo. One-half additional embryo, especially ifproven euploid, represents significant additional pregnancy potential inwomen with DOR, who usually produce only relative small embryo numbers.Indeed, this reflects approximately a one-third improvement in euploidembryo yield, and results in the availability of at least one embryo fortransfer in all post-DHEA cycles. In comparison, only 52.6% of untreatedcycles achieved the same goal. This is a statistically significantdifference in embryo transfers. Pretreatment with DHEA of women with DORsignificantly increases their chances for the transfer of at least oneeuploid embryo and may, therefore, at least in part, explain the higherpregnancy rates reported with DHEA supplementation.

Based on the incremental improvement in DHEA effects for up to fourmonths, and the correlation of the time span to a full cycle offollicular recruitment, it is suspect that DHEA may affect apoptoticprocesses during follicular recruitment. As a consequence, more healthyfollicles survive maturation, reach the stage of gonadotropinsensitivity and become subject to exogenous gonadotropin stimulation.These, in turn, also could be expected to have a higher probability ofeuploidy.

Increasing aneuploidy rates with female age are considered the principlecause of decreasing spontaneous female fertility, increasing infertilityand rising miscarriage rates. DHEA may improve euploidy rates as will bediscussed in more detail herein, and in turn, improves spontaneousfemale fertility, decreases the rate of female infertility and reducesmiscarriage rates.

Example 5 DHEA Substitution Improves Ovarian Function

In a further study, a case of probable 17,20-desmolase deficiency,resulting in abnormally low estradiol, DHEA, androstenedione andtestosterone levels, is presented in a woman with a clinical history of,initially, unexplained infertility and, later, prematurely agingovaries.

This patient started attempting conception in 1996, at age 33. Afterfailing to conceive for over one year, she was diagnosed withhypothyroidism and was placed on levoxyl. She, thereafter, remainedeuthyroid for the whole period described in this case report. Sheentered fertility treatment at a prominent medical school based programin Chicago, in August of 1997, where, now age 34, she failed threeclomiphene citrate cycles. No further treatment took place until alaparoscopy was performed in October of 1999, at a prominentAtlanta-based infertility center (where the couple had relocated to),revealing stage II endometriosis which was laser vaporized. Followingsurgery, a fourth clomiphene citrate cycle and a firstgonadotropin-stimulated cycle failed. Table 5 presents selected key labdata for all ovarian stimulation cycles the patient underwent. A firstin vitro fertilization (IVF) cycle was performed, at age 36, in Octoberof 2000.

This cycle resulted in expected oocyte and embryos yields. Three embryoswere transferred, resulting in a chemical pregnancy. Three other embryoswere cryopreserved. However, because of a persistently thin endometrium,a number of attempts at transfer were cancelled.

In April of 2001, the patient was, based on an abnormal glucosetolerance test, diagnosed with insulin resistance, and was placed onmetformin, 500 mg thrice daily. She had no signs of polycystic ovariandisease: her ovaries did not look polycystic, she was not overweight,had no signs of hirsutism or acne, and androgen, as well as estradiol,levels were in a low normal range (Table 2). In June of 2001 (age 37), asecond IVF cycle was initiated. In this cycle the patient demonstratedthe first evidence of ovarian resistance to stimulation in that sheproduced only six oocytes. Only one out of five mature oocytefertilized, despite the utilization of intracytoplasmic sperm injection(ICSI). The previously cryopreserved embryos were, therefore, thawed andtransferred, together with the one fresh embryo from the current cycle.The transfer was unsuccessful.

In August of 2001, the female's FSH level for the first time wasabnormally elevated (11.4 mIU/ml), with estradiol levels remaininglow-normal. Subsequent FSH levels were 19.1, 9.7 and 9.8 mIU/ml inNovember and December (twice), respectively, all with low-normalestradiol levels. FSH levels continued to fluctuate in 2002, with levelsreported as 11.4 mIUI/ml in February, 8.7 in March, 13.6 in June and19.6 in September, while estradiol levels remained persistentlylow-normal (Table 2).

A third IVF cycle was started in October of 2002, with a baseline FSH of11.3 mIUI. Ovarian stimulation, which in the prior two cycles had beengiven with only recombinant FSH (and antagonists), was now given in acombination of recombinant FSH and hMG at a combined dosage of 300 IUdaily. Estradiol levels reached only 890 pg/ml and only 5 oocytes wereretrieved. All four mature oocytes fertilized and four embryos weretransferred. A twin pregnancy was established by ultrasound and asingleton by heart beat. This pregnancy was, however, miscarried andconfirmed as aneuploid with a Trisomy 22.

The fact that this cycle, after the addition of hMG to the stimulationprotocol, appeared more successful, led the patient to a search of themedical literature. Like our previously reported patient (Barad andGleicher, 2005), this patient discovered a case series. The paperattracted the patient's interest. In follow up, she asked a medicalendocrinologist to evaluate her adrenal function. An initial evaluationrevealed very low DHEA, DHEA-S, androstenedione and testosterone levels(Table 2). An ACTH-stimulation test was ordered which showed theexpected increase in cortisol level, but unchanged, low DHEA, DHEA-S andtestosterone levels (Table 3). The patient was advised by her medicalendocrinologist that the most likely explanation for such a finding wasa 3-beta hydroxysteroid dehydrogenase deficiency. This enzyme defect is,however, associated with an accumulation of DHEA and, therefore, highlevels of the hormone. (Speroff et al., 1999a). Such a diagnosis for thepatients is, therefore, unlikely. Instead, abnormal 17,20-desmolase(P450c17) function would be expected to result in exactly the kind ofhormone profile, reported in this patient after ACTH stimulation,characterized by persistently low DHEA, androstenedione, testosteroneand estradiol levels, but normal aldosterone and cortisol levels.

In July of 2003, the patient was started on 25 mg daily of micronizedDHEA. After five weeks of treatment, DHEA, DHEA-S and androstenedionelevels had normalized into mid-ranges. (Even though androstenedione ispartially produced through the activity of 17,20-desmolase from17-hydroxyprogesterone, part is also derived from DHEA through theactivity of 3-beta hydroxysteroid dehydrogenase [Speroff et al., 1999a].The normalization of androstenedione, after DHEA administration,therefore, also speaks for an underlying 17,20-desmolase defect, and nota 3-beta hydroxysteroid dehydrogenase deficiency.) In the third andfourth month, following the start of DHEA supplementation, the patientovulated spontaneously with estradiol levels of 268 and 223 pg/ml (Table2), respectively, measured on the day of LH surge.

On Jan. 28, 2004 (age 39), and after DHEA therapy of approximately sixmonths, a fourth IVF cycle was initiated. Her baseline FSH level in thatcycle was 9.6 mIU/ml, estradiol 56 pg/ml. Stimulation took place with300 IU of recombinant FSH (without hMG) and with an agonist flareprotocol. Estradiol levels reached a peak of 1764 pg/ml, 8 oocytes wereretrieved, six out of seven mature oocytes fertilized and six embryoswere transferred. A triplet pregnancy was established with heart beats.Two, out of the three fetuses lost heart beat spontaneously, and thepatient delivered by cesarean section, at term, a healthy singleton maleinfant.

At surgery, her ovaries were closely inspected and described as “old”and “small”, with the left one being described as “almost dead.” DHEAand DHEA-S levels at six months of pregnancy were reported at “recordlows.” DHEA-S, six weeks post-delivery, was still very low (Table 5). Attime of this report, the male offspring is nine months old and themother has been re-started on DHEA in an attempt at another pregnancy.

DHEA substitution resulted in apparently normal peripheral DHEA levels,spontaneous ovulation and normal estradiol production by the ovaries. AnIVF cycle, after approximately six months of DHEA substitution, showed,in comparison to a pre-DHEA IVF cycle, improved peak estradiol levels,increased oocyte and embryo numbers and resulted, at age 39, after 6years of infertility therapy, in a triplet pregnancy and a normalsingleton delivery.

Low DHEA levels appear associated with female infertility and ovarianaging. DHEA substitution normalizes peripheral DHEA levels and appearsto improve ovarian response parameters to stimulation.

The reported patient exhibited some of the classical signs ofprematurely aging ovaries (Nikolaou and Templeton, 2003; Gleicher N.,2004) which include ovarian resistance to stimulation, poor egg andembryo quality and prematurely elevated FSH levels. The patient wasinitially thought to have largely unexplained infertility. She laterdeveloped quite obvious signs of prematurely aging ovaries and, finally,even showed elevated FSH levels.

It has been previously suggested that the decrease in DHEA levels, withadvancing female age, may be an inherent part of the ovarian agingprocess and may, at least in part, and on a temporary basis, be reversedby external DHEA substitution (Barad and Gleicher, 2005, 2005a). Thiscase demonstrates that low DHEA levels are, indeed, associated with allthe classical signs of both prematurely and normally aging ovaries.While association does not necessarily suggest causation, the observedsequence of events in this patient supports the notion that low DHEAlevels adversely affect ovarian function.

Once the patient was administered oral DHEA, a reversal of many findingscharacteristic of the aging ovary, were noted. First, the patient's DHEAand DHEA-S levels normalized. In subsequent natural cycles an apparentlynormal spontaneous follicular response was observed, with normalovulatory estradiol levels in a patient with persistently low estradiollevels before DHEA treatment (Table 5). The response to ovarianstimulation improved, quantitatively and qualitatively, as the patientimproved peak estradiol levels, oocyte and embryo numbers and, as thesuccessful pregnancy may suggest, also embryo quality.

One cannot preclude that other factors contributed. For example, theovarian stimulation protocol had switched from an antagonist to anagonist flare protocol. The data demonstrates that a maximal effect ofDHEA is achieved after at least about four consecutive months of use.This patient was on DHEA treatment for approximately six months beforeshe conceived the pregnancy that led to her first live birth.

This case is well documented in its DHEA deficiency and in its mostlikely cause. The reported adrenal response to ACTH stimulation (Table5) lends itself to the explanation (FIG. 1) of 17,20-desmolasedeficiency.

TABLE 5 Relevant laboratory results Date TEST RESULT (Normal values)*COMMENTS August 1997 TSH .8 mlU/l (0.4-5.5) Diagnosis of hypothyroidismMay 1999 FSH 4.0 mIU/ml April 2001 Glucose tolerance test Elevated ½hour insulin levels Diagnosis of Normal Glucose levels insulinresistance June 2001 FSH 7.7 mIU/ml Estradiol 33 pg/ml August 2001Testosterone free/ 2 ng/dl (3-29) weakly bound free only 1 pg/ml (1-21)total 13 ng/dl (15-70) DHEA-S 96 mcg/dl (12-379) Total Cortisol 14.2mcg/ml (4-22) FSH 11.4 mIU/ml Diagnosis of prem. ov. aging Estradiol 45pg/ml October 2001 Estradiol periovulatory 119 pg/ml November 2001Testosterone total 23 ng/ml (14-76) Androstenedione 98 ng/ml (65-270)Ovarian antibodies negative FSH 19.1 mIU/ml Estradiol 23 pg/ml December2001 FSH 9.7 mIU/ml Estradiol 27 pg/ml February 2002 Testosterone total<20 ng/dl (20-76) Androstenedione 76 ng/dl (65-270) FSH 11.4 mIU/mlEstradiol 28 pg.ml March 2002 Testosterone total 16 ng/dl (15-70) FSH8.7 mIU/ml Estradiol 29 pg/ml May 2002 FSH 13.6 mIU/ml Estradiol 30pg/ml Periovulatory 139 pg/ml June 2002 periovulatory 50 pg/ml September2002 Testosterone total 15 ng.dl (15-70) Free 1.6 pg/ml (1-8.5) % free0.0107 (0.5-1.8) Estradiol periovulatory 136 pg/ml October 2002 FSH 11.3mIUI/ml Estradiol 43 pg/ml February 2003 FSH 13.6 mIU/ml Estradiol 33pg/ml March 2003 FSH 8.9 mIU/ml Estradiol 67 pg/ml May 2003 Anti-adrenalantibodies negative Estradiol periovulatory 139 pg/ml DHEA 132 ng/dl(130-980) DHEA-S 79 mcg/dl (52-400) Testosterone total 34 ng/dl (20-76)Free 3 pg/ml (1-21) July 2003 DHEA TREATMENT START DHEA 296 ng/dl(130-980) DHEA-S 366 mcg/dl (52-400) Androstenedione 121 ng/dl (65-270)September 2003 Estradiol periovulatory 268 pg/ml October 2003 FSH 14.7mIUI/ml Estradiol 44 pg/ml Periovulatory 224 pg/ml November 2003 FSH 17mIU/ml Estradiol 38 pg/ml December 2003 DHEA 278 ng/ml (130-980) DHEA-S270 mcg/dl (52-400) Testosterone total 25 ng/ml (20-76) free and weeklybound 4 ng/dl (3-29) free 2 pg/ml (1-21) January 2004 FSH 18 mIU/ml FSH9.6 mIU/ml 4th IVF Estradiol 56 pg/ml CYCLE START August 2004MID_PREGNANCY DHEA 74 ng/dl (135-810) DHEA-S 27 mcg/dl(**) October 2004DELIVERY December 2004 DHEA-S 52 mcg/dl (44-352) *Laboratory tests wereperformed at varying laboratories (**)No pregnancy levels available fromlaboratory

TABLE 6 ACTH stimulation test HORMONE BASELINE +30 MINUTES +60 MINUTESDHEA-S (mcg/ml) 87 88 83 Cortisol total (mcg/dl) 15 26 27 Testosteronetotal (ng/dl) 28 32 33 free and weakly bound 5 5 5 free 3 3 3

This case report presents further evidence for DHEA deficiency as acause of female infertility and as a possible causative agent in theaging processes of the ovary. It also presents further confirmation ofthe value of DHEA substitution whenever the suspicion exists thatovaries may be lacking of DHEA substrate. Finally, this case reportraises the important question what the incidence of adrenal17,20-desmolase (P450c17) deficiency is in women with prematurely agingovaries.

Example 6 Increase Male Fetus Sex Ratio

Androgenization of females with dehydroepiandrosterone (DHEA), as werecently have been utilizing in the fertility treatment of women withdiminished ovarian reserve, in combination with the investigation ofspontaneous, versus in vitro fertilization (IVF) —conceived, pregnanciesallows for an investigation of the basic theory of sex allocation andits possible pathophysiologic mechanisms.

The treatment protocol for long-term supplementation with DHEA that mayimprove oocyte and embryo quantity, quality, pregnancy rates and time toconception in women with diminished ovarian reserve, involves 25 mg ofmicronized, pharmaceutical grade DHEA, TID will usually uniformly raiselevels of unconjugated DHEA above about 350 ng/dl, and, therefore, raisebaseline testosterone. Estradiol baseline levels may also be raised.

A retroactive review of either ongoing or delivered pregnancies beyond20 weeks gestational age, conceived while on DHEA treatment for at least60 days, revealed 23 women. A total of 19 pregnancies were recorded with16 singleton and 3 twin pregnancies. The medical records of all 19 womenwere reviewed in order to determine whether they conceivedspontaneously, defined as including pregnancies conceived withintrauterine inseminations, or by IVF. If conception had occurred byIVF, it was recorded whether fertilization was spontaneous or byintracytoplasmic sperm injection (ICSI).

As a control group, seven women were selected who had undergone one IVFcycle with preimplantation genetic diagnosis (PGD), while for at least60 days on DHEA supplementation, but had not conceived. The PGD data,defining each embryo's gender, were recorded. Statistics were performedusing a binomial runs test, comparing seen distributions with anexpected distribution of 50 percent, with p<0.05 defining significance.

Sixteen singleton pregnancies resulted in 11 males and 5 females (N.S.).Two of three twin pregnancies were heterozygous and one homozygous. Ifoutcomes of both heterozygous twins, but of only one homozygous twin,were added, the final gender distribution was 15 males and 6 females(p=0.078, N.S.)

Amongst six pregnancies, spontaneously conceived, the distributionbetween female and male offspring was equal, at three and three,respectively. Whereas amongst the remaining 15 offspring, which wereproducts of pregnancies achieved through IVF, the distribution was 12males and 3 females (p=0.035). Only one IVF patient failed to have ICSI.Amongst women undergoing IVF and PGD, 53 embryos were analyzed from 17IVF cycles, all having undergone ICSI. The gender distribution was notsignificantly skewed, with 27 being male and 26 female.

This study allows for the dissection of the conception process into itsvarious stages and, therefore, permits an analysis of, not only thebasic question whether androgenization does indeed, affect genderselection in the human, but also how such a selection may be influenced.

The here presented data, demonstrating a strong trend towardssignificance overall, and significance (p=0.035) amongst IVF patients,suggest, convincingly that gender determination may be influenced byhormonal environment. Assuming an effect of androgens on genderselection, such women should give birth to a preponderance of maleoffspring. Confirming such a finding could present a potentialadditional explanation for the evolutionary preservation of PCOS inpractically all human races.

Example 7 Increase Pregnancy Rates

In an additional study, a retrospective analysis of a 190 women withdiminished ovarian function above 30 years old, who were treated between1999 and December 2005 was completed to assess the impact of DHEAsupplementation on the time interval to the establishment of pregnancy.

A prequalification for each patient's diagnosis of diminished ovarianfunction was either a sub-diagnosis of premature ovarian aging (POA) ora sub-diagnosis of diminished ovarian reserve (DOR). POA was, in turn,defined as baseline follicle stimulating hormone (b-FSH), on Day 2/3 ofa cycle as <12 mIU/ml, but exceeding the 95% CI of the mean value forthe patient's age group.

Specifically, this meant b-FSH.gtoreq.7.4 mIU/ml at age 30-34 years and.gtoreq.8.6 mIU/ml at age.gtoreq.35 years. DOR, in turn, was defined asb-FSH.gtoreq.12 mIU/ml and/or a baseline estradiol level.gtoreq.75pg/ml. 49 patients were confirmed with POA and 52 patients wereconfirmed with DOR, creating a control group of 101 women. Because ofpotential impending loss of ovarian function in the control group women,the control group women were treated with IVF as soon as possible.

During the time studied, the study group consisted of 89 patients, withdiminished ovarian function (POA 24, DOR 65). Each person in the studygroup was placed on DHEA supplementation. The DHEA supplementationincluded administering about 25 mg of (pharmaceutical grade) micronizedDHEA, three times daily, for up to about four months (mean 3.8±0.3months). In contrast to the control group, women in the study group didnot enter IVF right away. This delay of IVF treatment allowed thepossibility of spontaneously conceived pregnancies. Those patients whodid not conceive spontaneously within four months of beginning DHEAentered IVF.

Methods of Example 7

Ovarian stimulation was identical for study and control groups andcomprised microdose agonist flare, followed by maximal dosagegonadotropin stimulation, using about 300-450 IU of FSH and about 150 IUof HMG. Study patients received DHEA continuously until a positivepregnancy test was obtained or until the patient dropped out oftreatment. DHEA and DHEAS levels were monitored monthly, and patientswere interviewed at each visit about adverse reactions to DHEAsupplementation. Because of the dynamics of the DHEA treatmentalgorithm, at the time of this data analysis, 16 women in the studygroup were at various stages of DHEA supplementation, prior to anyintervention, 9 women received ovarian stimulation while on DHEA, and 64have undergone an IVF cycle.

In order to assess the impact of DHEA supplementation on time intervalto the establishment of pregnancy, this study was designed as alife-table analysis, measuring not only total pregnancy rates but alsothe time between initial presentation and end of last treatmentintervention.

Each recorded clinical pregnancy, defined as positive fetal cardiacactivity on ultrasound examination after 6 weeks, was recorded as apositive outcome. Patients who continued treatments beyond the studyperiod or stopped treatments were considered right censored data at theend of the study period, or at treatment cessation, respectively.

The following factors were compared between study and control groups:female age, months of infertility prior to initial visit, length oftreatment from first presentation, gravidity, race, IVF treatments,maximal baseline FSH levels, maximal baseline estradiol levels, IVFcycle cancellation rates, oocyte numbers, number of embryos transferred,implantation rates, cumulative clinical pregnancy rates and miscarriagerates.

A Cox regression analysis was used to evaluate time-to-event. The modelthat we used stratified for level of ovarian reserve (POA and DOR) andadjusted for age, day 3 FSH, fertility treatments (none, IntrauterineInsemination and controlled ovarian hyperstimulation (IUI/COH), or IVF)and race/ethnicity. A trend in pregnancy rates over months of DHEAexposure with an interaction term for time and DHEA months of exposurewas tested. SPSS for Windows, Standard version 10.0.7 (SPSS Co. Chicago,Ill.) was utilized for data analysis. Continuous outcomes are presentedas mean±1 standard error. Univariate comparisons were made with analysisof variance, or by using Fisher's exact test.

Results of Example 7

Table 7 summarizes patient characteristics. As can be seen, studypatients were slightly older than the controls at 41.6±0.4 and 40.0±0.4years (p<0.05) respectively. Pregnancy histories, duration ofinfertility and of treatment (in months) were similar between the twogroups. Controls represented a non significant larger proportion ofminorities, received more treatment cycles overall (1.6±0.9 versus1.3±1.0; p<0.05) and differed significantly in the various treatmentsthey received (p<0.001). Study patients demonstrated a non-significantlyhigher b-FSH 16.0±1.2 13.6±1.0 mIU/ml) and a significantly higherbaseline estradiol level (366±53 versus 188±24 pml/ml; p<0.05). Morewomen in the study group had b-FSH.gtoreq.10 mIU/ml that amongstcontrols (73% versus 51.5%; p<0.05). In addition, greater proportion ofwomen in the study group had DOR (p<0.005).

TABLE 7 Characteristics of DHEA Treated and Controls DHEA Control p N 89101 Age 41.6 ± 0.4 40.0 ± 0.4 <0.05 Months Infertility 44.5 ± 4.8 41.9 ±5.9 ns Months from First Visit  8.1 ± 0.7  7.8 ± 1.0 ns Race ns White 62(70.5%) 57 (56.4%) — Hispanic 7 (7.9%) 12 (11.9%) — Black  9 (10.2%) 14(13.9%) — Asian 11 (12.5%) 18 (17.8%) — Cycles of Treatment 1.3 ± 1  1.6 ± 0.9 <0.05 Treatment <0.01 No Treatment 16 (18.2%) 0 (0%)   —IUI/COH  9 (10.2%) 0 (0%)   — IVF 64 (71.6%) 101 (100%)   — Day 3 FSH(mIU/ml) 16.0 ± 1.2 13.6 ± 1.0 ns Day 3 E2 (pmol/ml) 366 ± 53 188 ± 24<0.05 Ovarian Function  <0.005 POA 24 (27%)   49 (48.5%) — DOR 65(73%)   52 (51.5%) —

Table 8 lists the results of univariate comparisons of treatmentoutcomes. As can be seen, confirming a more severe degree of diminishedovarian function, the study group produced significantly fewer oocytes,normal day-3 embryos (2.4±0.03 versus 3.5±0.2; p<0.05) and transferredembryos (2.1±0.2 versus 2.7±0.2; p<0.05). Cycle cancellations were,however, nominally higher among the controls (25.7% versus 14.3%).

TABLE 8 Univariate Comparison of Results Between Control and DHEATreated Patients DHEA Control p N total; (IVF) 89; (64) 101 Months ofDHEA 3.8 ± 0.3 — — Cancellation (IVF)  9/63 (14.3%) 26/101 (25.7%) nsOocytes 3.9 ± 0.4 5.8 ± 0.5 <0.01 Normal Day 3 embryos 2.4 ± 0.3 3.5 ±0.2 <0.05 Transferred embryos 2.1 ± 0.2 2.7 ± 0.2 <0.05 Positive hCG(>25 mIU/ml) 26/86 (30%)  18/101 (18%)   ns Implantation (FH/Embryos13/101 (11.4%) 11/148 (6.9%)  ns trans) Clinical Pregnancy  25/89(28.1%) 11/101 (10.9%) <0.01 No Treatment  6/16 (35.3%) — — IUI/COH  6/9 (68.7%) — — IVF  13/64 (20.6%) 11/101 (11.9%) ns Miscarriage (Perclinical  5/25 (20%)  4/11 (38%) ns Pregnancy)

Overall clinical pregnancy rates were significantly higher in studypatients (28.1% versus 10.9%; p<0.01). Remarkably, almost one-half ofall pregnancies in the study group were established spontaneously beforeIVF start; however, even within the patients reaching IVF, there was astrong trend towards higher pregnancy rates (20.6% versus 11.9%).

Approximately two months after initiation of treatment the mean DHEA andDHEAS levels at cycle day 2 blood drawing were in the low normal ranges.Few patients reported side effects from DHEA use. These included mildtransient acne on the face, chest or back, oily skin and mild hair loss.No facial or body hair growth was reported, nor was there any deepeningof voice. Some patients reported an increased sense of well-being orincreased libido.

Cox regression of months from initial visit until clinical pregnancy,adjusted for age, race/ethnicity, fertility treatment, and stratifiedfor level of ovarian reserve (POA and DOR), revealed that DHEA treatedpatients had a significantly increased proportional hazards ratio forclinical pregnancy relative to controls (HR 3.8; 95% CI 1.2 to 11.8;p<0.05). FIGS. 6 and 7 show proportional hazard curves of clinicalpregnancy by months from their initial visit. Specifically,

FIG. 6 is a graph showing cumulative pregnancy rate of time from initialvisit to clinical pregnancy or censor by DHEA for women with prematureovarian aging, and FIG. 7 is a graph showing cumulative pregnancy rateof time from initial visit to clinical pregnancy or censor by DHEA forwomen with diminished ovarian reserve. The curves reveal a rapidlyseparating increase in cumulative clinical pregnancies between study andcontrol groups from the first month on.

Extended Cox models with correction for time dependent variables “monthsof DHEA use” and “Treatment” did not decrease the proportional hazardsestimation of pregnancy associated with DHEA treatment (HR 4.8; 95% CI1.6 to 14.2; p=0.005).

Discussion of Example 7

A significantly increased pregnancy rate in a group of women with a verypoor prognosis for pregnancy has been determined. A strength of thisstudy is its rather large sample size.

Spontaneous background pregnancy rates in average infertile women occurat an approximate rate of one to two percent per month. Spontaneouspregnancies in women with clear evidence of diminished ovarian functionare obviously an even rarer occurrence. Given the degree of loss ofovarian reserve in this group, a 28.1% cumulative pregnancy rate in apatient population, previously largely referred into oocyte donation, isquite remarkable.

DHEA supplementation can improve ovarian function in women withdiminished ovarian reserve. Study and control patients receivedidentical ovarian stimulation protocols during IVF cycles. IVF protocolsduring the study years 1999-2005 did not significantly change duringthis time. Specifically, the protocols may include administeringmicrodose agonist/gonadotropin stimulations in women with diminishedovarian reserve.

The mechanism of DHEA's action on the ovary remains speculative. DHEAdeclines with age and DHEA supplementation may simply improve thesubstrate pool for steroidogenesis, since DHEA is a precursor hormonefor estradiol and testosterone.

Androgens may, however, influence ovarian follicular growth not only byacting as metabolic precursors for steroid production, but also byserving as ligands for androgen receptors or by other, non-classicalmechanisms. During ovulation induction with exogenous gonadotropins,DHEA is the prehormone for up to 48% of follicular fluid testosterone,which is, in turn, the prehormone for estradiol. There is evidence thatandrogens act, together with FSH, to stimulate folliculardifferentiation. Androgens are also known to promote steroidogenesis andfollicular recruitment and to increase insulin-like growth factor(IGF-1) in the primate ovary. DHEA-treated rat ovaries express elevatedlevels of IGF-1 in pre-antral and early antral follicles.

A transient increase in IGF-1 in patients undergoing exogenousgonadotropin ovulation induction after pretreatment for only eight weeksof DHEA has previously been reported and it was hypothesized that theeffect of DHEA on ovulation induction might have been mediated byincreased IGF-1.

Higher baseline testosterone levels have been associated with improvedIVF outcomes, and higher serum testosterone has been correlated withhigher oocyte numbers retrieved at IVF. Some authors have suggested thatimproved outcomes in women with diminished ovarian reserve afterco-treatment with aromatase inhibitors may be the consequence ofinduction of FSH receptors on granulosa cell by androgens. The resultantovarian response may then lead to improved follicular survival,increased follicle numbers and higher estradiol levels duringstimulation, as classically also observed in polycystic ovarian disease.

Human polycystic ovaries have been described as representing a“stock-piling” of primary follicles, secondary to an alteration at thetransition from primordial to primary follicle. It is possible that DHEAtreatment may create PCO like characteristics in the aging ovary. Longterm exogenous androgen exposure can induce PCO-like histological andsonographic changes. Androgens have been reported to suppress apoptosis.Exogenous DHEA exposure may occur during the first two weeks ofpregnancy.

In summary, a significant increase in the odds of pregnancy among DHEAtreated women has been determined. This increase appears to be rapid inonset and to continue progressively within eight months of initialobservation.

Example 8 Decrease Miscarriage Rates

In a further study, women (i.e, women with progressively decliningovarian function) with diminished ovarian reserve were administered DHEAto assess the effect of DHEA on miscarriage rates.

Since women with diminished ovarian reserve produce only few oocytes andembryos, preimplantation genetic diagnosis (PGD) in association with IVFis only rarely indicated, and, indeed, may be detrimental. To accumulatedirect ploidy data on a large enough statistical patient sample is,therefore, difficult. Because spontaneous miscarriage rates arereflective of aneuploidy rates, the study presented herein includespregnancy outcomes after DHEA supplementation from two independent NorthAmerican fertility centers and compares those with age-specific nationaloutcome data after IVF.

Materials and Methods of Example 8

Based on reported clinical experiences, the indications for DHEAsupplementation have changed over the years, with women above age 40since 2007 receiving routine supplementation, and younger womenreceiving supplementation only selectively. This means that under age 40women receive supplementation only if they demonstrate elevatedage-specific baseline follicle stimulating hormone (FSH) levels and havedemonstrated in at least one cycle inappropriately low oocyte yield within vitro fertilization (IVF) following standard ovarian stimulation withgonadotropins.

DHEA supplementation involves the use of pharmaceutical grade micronizedDHEA at a dosage of about 25 mg, three times daily. Patients are on DHEAsupplementation for at least about two months prior to oocyte retrieval.This period of minimal pretreatment is based on the recognition that attwo months pregnancy curves between DHEA pretreated and control patientsstatistically diverge. DHEA is maintained until pregnancy is establishedand is discontinued with positive pregnancy test.

Toronto West Fertility Associates, in Toronto, Canada, started utilizingDHEA independent of the use of DHEA at the Center for Human Reproduction(CHR) in New York, N.Y. In December of 2007, Toronto's medical directorforwarded a detailed electronic record of the center's all-inclusiveDHEA experience for analysis to CHR. This study, therefore, reports onthe miscarriage rate of pregnancies, independently established underDHEA supplementation at both fertility centers, and compares theserates, age-stratified, to miscarriage rates reported in a national IVFdata base in the U.S. for the year 2004. The definitions of clinicalpregnancy, and of miscarriage, used herein follow the reportingrequirements of this national data base, defining a clinical pregnancyas a pregnancy, confirmed by ultrasound examination.

It is important to note that DHEA supplemented patients universallysuffered from severely diminished ovarian reserve. Their pregnancyexpectations were, therefore limited. Patients who conceived a clinicalpregnancy, thus, represented only a minority of DHEA supplementedpatients at both centers.

Miscarriage rates of DHEA supplemented patients were compared withnational IVF outcome statistics, reported annually under Federal mandateby the Centers for Disease Control. The data utilized for this studyreflect 2004 United States national statistics. Pregnancy andmiscarriage rates at the two centers were pooled after confirmation ofhomogeneity of variance. Common odds ratios of the pooled miscarriagerates among age stratified pregnant patients were compared between thepooled rates and the 2004 US national rates utilizing theMantel-Haenszel common odds ratio. Statistical analyses were performedusing SPSS Windows, standard version 15.0.

Results of Example 8

New York reported 40 and Toronto 33 DHEA pregnancies, for a combinedDHEA pregnancy experience of 73 pregnancies. New York reported sixmiscarriages, for a clinical miscarriage rate of 15.0%, and Torontoreported five miscarriages, for a clinical miscarriage rate of 15.2%,for a combined miscarriage rate of 11/73 (15.1%). For analysis, the 2004miscarriage rate in the national U.S. registry of 17.6% was used.

As seen in Table 9 (below) and FIG. 8, miscarriage rates after DHEAsupplementation, stratified for age, were lower at all ages [OR 0.49(0.25-0.94; p=0.04)]. The decrease in miscarriage rate was, however,especially apparent above age 35 years.

TABLE 9 Age-stratified pregnancy and miscarriage rates Age (years) <3535-37 38-40 41-42 >42 DHEA Pregnancies NY 10 5 6 10 9 TO 7 10 13 0 3Miscarriages NY 1 0 0 2 3 TO 1 1 3 0 0 Misc. Rate (%) NY 10.0 0.0 0.020.0 33.3 TO 14.3 10.0 23.1 — 0.0 TOTAL 11.8 6.7 15.8 20.0 25.0 (±95%CI) (15.0) (13.0) (16.0) (25.0) (25.0) NATIONAL Misc. Rate (%) 14.0 17.123.1 36.6 50.1 (±95% CI) (1.0) (1.0) (1.0) (2.0) (5.0) Decrease in Misc.Rate −15.7 −60.8 −31.6 −45.3 −50.1 with DHEA (%) Miscarriage rates afterDHEA supplementation, stratified for age, were lower at all ages [OR0.49 (0.25-0.94; p = 0.04)]. The decrease in miscarriage rate was,however, especially apparent above age 35 years. NY - Center for HumanReproduction, New York; TO - Toronto West Fertility Associates, Toronto,Canada

Discussion of Example 8

The data reported herein demonstrate a significantly diminishedmiscarriage rate in women with diminished ovarian reserve, in comparisonto a standard IVF population, if pretreated for at least two months withDHEA. Specifically, as shown in Table 9, the percentage decrease inmiscarriage rate with DHEA supplementation for women with diminishedovarian reserve under the age of 35 was 15.7, for women between the agesof 35-37 was 60.8, for women between the ages of 38-40 was 31.6, forwomen between 41-42 was 45.3, and for women above the age of 42 was50.1. This effect appears particularly pronounced above age 35 years.

This is a remarkable observation that is further strengthened by thefact that, due to their severely diminished ovarian reserve, the studiedDHEA supplemented women represent a highly unfavorable patientpopulation. It has been reported that women with diminished ovarianreserve experience exceedingly high miscarriage rates, far in excess ofstandard IVF patients with normal ovarian reserve. For example,miscarriage rates of 57.1 percent under age 35 in women with diminishedovarian reserve, 63.5 percent between ages 35 and 40 in women withdiminished ovarian reserve, and as high as 90 percent above age 40 yearsin women with diminished ovarian reserve have been reported. Consideringthe fact that national U.S. IVF data represents only a minority of womenwith diminished ovarian reserve, the finding that DHEA supplementationsignificantly reduced miscarriage rates in all age groups below those ofan average national IVF population is remarkable.

While on first glance the larger degree of reduction in miscarriage ratein older women may surprise, it should not. Aneuploidy rates increasewith age and, indeed, age 35 is generally considered the age cut off,where more aggressive prenatal genetic screening becomes indicated. IfDHEA affects aneuploidy rates, then one would, indeed, expect a muchlarger beneficial effect after, rather than before, age 35, becauseolder women usually produce fewer embryos, and the relative benefit froma decrease in aneuploidy rate on the number of euploid embryostransferred in IVF will, therefore, increase with advancing female age.

Aneuploidy is a frequent finding even in young women. As women age, theprevalence of aneuploidy increases further, at times reaching close to90 percent in women above age 40. Interestingly, women who demonstrateclinical evidence of prematurely aging ovaries do not also demonstrateprematurely enhanced aneuploidy rates. They maintain the expectedage-specific aneuploidy, dictated by their chronological age, andtherefore, experience similar implantation—and pregnancy rates, though,because of decreased oocyte and embryo numbers, reduced cumulativepregnancy rates. It, therefore, should not surprise that women under age35, even though suffering from a significant degree of prematurelydiminished ovarian reserve, did not benefit as much from DHEA as olderwomen.

This study demonstrates a statistical association between DHEAsupplementation and decreased miscarriage rates. The reported dataoffers enough circumstantial evidence to suggest that DHEA bothdecreases miscarriage rates and reduces aneuploidy rates in humanembryos.

The presented data helps to explain why DHEA supplementation increasesegg and embryo quality, improves pregnancy rates and speeds up time toconception. Egg and embryo quality is, of course, at least partially areflection of ploidy. Embryos with less aneuploidy can be expected tolead to more pregnancies, resulting in more, and quicker, conceptions.

The concept of embryo selection by improving ploidy has been the basisfor attempts at improving pregnancy rates and reducing miscarriage ratesvia preimplantation genetic screening (PGS). The utility of PGS hasrecently, however, been seriously questioned since, especially in womenwith only few embryos, the necessary embryo biopsy may cause more harmto pregnancy chances than the potential benefits, derived from embryoselection offer. DHEA supplementation, therefore, may represent a muchsimpler, more cost effective and, most importantly, safer method ofembryo selection for ploidy than PGS.

It should not be overlooked that the here presented study addresses onlyinfertile women with a significant degree of diminished ovarian reserve.As already noted, they represent a very unfavorable patient population,with exceedingly high expected miscarriage rates. However, even thoughinfertile women with normal ovarian reserve have significantly lowermiscarriage rates, they in general still experience higher miscarriagerates than the average population. While the here-reported miscarriagerates in DHEA patients are remarkably low, caution should, therefore, beexercised in concluding automatically that the observed DHEA effect canbe extrapolated to a general population. It is, however, quiteremarkable that the here-reported miscarriage rates in women withseverely diminished ovarian reserve at both study centers, stratified byage, were practically identical to those reported for the generalpopulation.

Based on the hypothesis that congression failure (gross disturbances inchromosome alignment on the meiotic spindle of oocytes) results from thecomplex interplay of signals regulating folliculogenesis (thusincreasing the risk of non-disjunction errors), it has been suggestedthat it may be possible to develop prophylactic treatments that canreduce the risk of age-related aneuploidy. DHEA may, indeed, be a firstsuch drug.

Assuming such a more universal effect of DHEA supplementation onaneuploidy rates, supplementation should also be investigated forinfertile women in general and, maybe, even for normally fertile womenabove age 35, who could receive DHEA as a routine preconceptionsupplement, akin to prenatal vitamins. Should efficacy of DHEAsupplementation in such a general population be proven, the potentialsignificance of such a finding on public health could be considerable.

As stated herein, and supported at least by the examples herein, DHEAsupplementation for at least two months increases egg numbers and eggquality and, therefore, also embryo numbers and quality. DHEA alsoimproves spontaneous pregnancy rates, IVF pregnancy rates, cumulativepregnancy rates and time to conception in prognostically otherwisehighly unfavorable patients. Further, DHEA statistically reducesmiscarriage rates, probably, at least partially, by reducing aneuploidyrates. Moreover, DHEA probably also increases the male/female birthratio. The effects of DHEA increase over time, reaching peaks afterapproximately four to five months of supplementation. It is suggestedthat the peak occurs at four to five months because this time period issimilar to the time period of a complete follicular recruitment cycle.

Example 8 Continued Background

Dehydroepiandrosterone (DHEA) supplementation may improve selectedaspects of ovarian function in women with diminished ovarian reserve.

DHEA supplementation improves response to ovarian stimulation withgonadotropins by increasing oocyte yield and embryo numbers. DHEAeffects increase over time, reaching peaks after approximately four tofive months of supplementation. DHEA, however, also increases oocyte andembryo quality, spontaneous pregnancy rates in prognostically otherwisehighly unfavorable patients on no further active treatments, pregnancyrates with in vitro fertilization (IVF), time to pregnancy andcumulative pregnancy rates.

DHEA may effect insulin-like growth factors (IGF-1) —mediated. On theother hand, because DHEA effects peak at four to five months, a timeperiod similar to the complete follicular recruitment cycle, we havespeculated that DHEA may effect follicular recruitment, possiblymediated via suppressive effects on apoptosis. Additionally, DHEA mayreduce aneuploidy in embryos.

Since approximately 80 percent of spontaneous pregnancy loss is theconsequence of chromosomal abnormalities, reduced aneuploidy should alsoreduce miscarriage rates. As women get older, and ovarian functionprogressively declines, miscarriage rates rise because of increasinganeuploidy. If DHEA, indeed, were to beneficially affect ploidy, DHEAsupplementation should, as an additional benefit in older women withseverely diminished ovarian reserve, therefore, result in reducedmiscarriage rates.

Since women with diminished ovarian reserve produce only small oocyteand embryo numbers with IVF, preimplantation genetic diagnosis (PGD) inassociation with IVF is only rarely indicated, and, indeed, may bedetrimental. To accumulate direct embryo ploidy data in such patientsis, therefore, difficult. Seeking alternatives, we were attracted by thefact that spontaneous miscarriage rates to such a large degree reflectaneuploidy rates. This study, therefore, presents pregnancy outcomesafter DHEA supplementation from two independent North American fertilitycenters and compares those with age-specific national USA outcome dataafter IVF.

Methods

DHEA supplementation: After approval by the center's institutionalreview board, the Center for Human Reproduction (CHR) in New York Cityhas been utilizing DHEA supplementation in women with diminished ovarianreserve since 2004. Based on reported clinical experiences, theindications for such supplementation have changed over the years: Ininitial stages, only older women, above age 42, were supplemented andonly if they had failed at least one IVF cycle and less than 4 oocyteshad been retrieved in confirmation of ovarian resistance to stimulation.By mid-2005, indications were expanded to all women above age 40 withevidence of ovarian resistance and a history of one failed prior IVFcycle. By early 2006 indications were further expanded to women underage 40 if they demonstrated elevated baseline follicle stimulatinghormone (FSH) levels above 10 mIU/ml and had shown ovarian resistance inat least one failed IVF cycle. By mid-2006 FSH baseline criteria werechanged from absolute FSH elevations to elevations in age-specific FSHlevels. All women above age 40 have been offered routine supplementationsince January 2007, while younger women, under age 40, are continuing tobe only selectively supplemented if demonstrating elevated age-specificbaseline follicle stimulating hormone (FSH) levels and, as previouslyreported, inappropriately low oocyte yield in at least one IVF cycle.

DHEA supplementation in all patients involves oral, pharmaceutical grademicronized medication at a dosage of 25 mg, three times daily (TID).Only morbidly obese women receive an increased daily dosage of 100 mgand no such women were involved in this study. This supplementationdosage was chosen and is continued to be used since DHEA use ahs shownto result in only minor side effects. Limited patient volume and fundingsources have prevented dose response studies and 25 mg DHEA TID dailyhas, therefore, remained the only standard treatment dosage. Patientsreceive at least two months of DHEA supplementation prior to oocyteretrieval, unless they conceive spontaneously during that time period.This minimum pretreatment period is based on the recognition that at twomonths pregnancy curves between DHEA pretreated and control patientsstatistically diverge. DHEA is maintained until pregnancy, and isdiscontinued with second positive pregnancy test.

Collaboration between centers: The utilization of DHEA at the Torontobased center was independently initiated, after that center's medicaldirector (E.R.) at a lecture (by N.G.) learned about the New Yorkcenter's DHEA experience. Toronto's data accumulation was unknown to theNew York center until in December of 2007, unsolicited, a detailedelectronic record of Toronto's DHEA experience was forwarded to New Yorkwith a request for combined analysis. The Canadian data were sequesteredto the New York center's confidential research data base, which isrestricted to one computer. Confidentiality and anonymity of submittedrecords was, therefore, maintained.

Control population: This study reports on miscarriage rates, at bothfertility centers, independently established under DHEA supplementation,and compares these rates, age-stratified, to miscarriage rates reportedin a national USA IVF outcome data base, which involves unselectedinfertility patients. While study populations at the New York andToronto centers, thus, involve women with significantly DOR, thenational control data reflect only a rather small percentage of womenwith this primary diagnosis.

DOR patients have in the past resisted prospective randomization. Tworegistered prospectively randomized, placebo controlled trials, one innew York City and a second in Europe, had to be abandoned for lack ofenrollments (Gleicher N and Barad D H, Unpublished data, 2006 and 2007).In the absence of such prospectively controlled studies, the questionarose how to establish statistically valid controls for observedmiscarriage rates: A control population should involve infertile womenunder treatment. It also should have maximal size, vary in agedistribution (to facilitate age stratification) and be all encompassing(to avoid selection biases). Since here presented DHEA data weregenerated in North America, a USA-based data base, fulfilling all ofthese criteria, was chosen.

The literature does not offer a unified definition of DOR. We define allwomen above age 40 years to suffer from DOR. In women under age 40 thediagnosis is only reached if age-specific ovarian function parameters.

Definitions of clinical pregnancy and of miscarriage follow thereporting requirements of this national data base, defining clinicalpregnancy, as confirmed by ultrasound.

Since patients at both study centers, as a prerequisite to DHEAsupplementation, had to suffer from DOR, their expectation of pregnancysuccess is very limited. Even considering a higher conception rate insuch patients after supplementation with DHEA, conceptions will occur inonly a small minority of DHEA supplemented cycles. The here reportednumber of consecutive pregnancies, therefore, represents a range ofapproximately 450 to 570 initiated DHEA treatment cycles.

Statistics: Miscarriage rates of DHEA supplemented patients werestatistically compared with national IVF outcomes, reported annuallyunder federal mandate by the Centers for Disease Control and Prevention,U.S. Department of Health and Human Services. The data utilized ascontrols for this study reflect 2004 United States IVF statistics,report cycle numbers, pregnancy percentages and live birth percentages,stratified for age. These detailed national data allowed calculation ofnumber of clinical pregnancies and number of live births for each agegroup, since neither is offered in the original data set. We thensubtracted live births from pregnancies, to derive number of failedpregnancies (i.e., all failed pregnancies were for purpose of this studyconsidered miscarriages) overall, and in each age category. Counts ofpregnancies and miscarriages were then entered into a series of two bytwo tables, stratified by age, and using the cross tabulation module ofSPSS 15.00.

Pregnancy and miscarriage rates at both fertility centers were pooledafter confirmation of homogeneity of variance. Common odds ratios of thepooled miscarriage rates among age stratified pregnant patients werecompared between the pooled centers and 2004 national rates, utilizingthe Mantel-Hanszel common odds ratio (tests for homogeneity of the oddsratio across layers were not significant, meeting assumption for use ofthis test) and using dichotomous exposure (DHEA versus controls) anddichotomous outcomes (live births versus spontaneous miscarriages),stratified by five age categories.

A secondary statistical analysis of the data was performed, byrecalculating for all five investigated age groups (<35, 35-37, 38-40,40-42 and >42 years) expected miscarriage rates for both patient groups,equalized for size. Both statistical analyses are presented in sequenceand were performed using SPSS Windows, standard version 15.0.

Institutional Review Board: The investigation of DHEA in women with DORhas been repeatedly approved by the center's Institutional Review Board(IRB). Since the here reported study only involved the evaluation of(electronic) medical records, and maintained their confidentiality, thehere presented study, based on a patient consent signed at time ofinitial registration, did not require further IRB approval. Aconfirmatory written statement from the chairman of the IRB is availableupon request.

Results and Discussion

New York reported 40 and Toronto 33 DHEA pregnancies, for a combinedDHEA pregnancy experience of 73 pregnancies. Among those pregnancies,New York registered six and Toronto five miscarriages, for clinicalmiscarriage rates of 15.0% and 15.2%, respectively, and a combinedmiscarriage rate of 11/73 (15.1%). In comparison, the total 2004miscarriage rate in the national USA registry was 17.6%. The odds ratioand 95% confidence interval (CI), stratified for age, that a woman wouldmiscarry was, thus, statistically significantly lower after DHEAsupplementation [OR 0.49 (0.25-0.94; p=0.04), suggesting a reduction inmiscarriage risk of approximately 50 percent (data not shown;Mantel-Hanszel, distributed as Chi-square with one degree of freedom,4.285; p=0.038).

When expected miscarriage rates were compared in both patient groups,equalized for number of patients, women after DHEA supplementationdemonstrated even more significant reductions in miscarriage rate(p<0.0001) suggesting an almost 80% reduction in miscarriage risk (datanot shown; Mantel-Haenszel, distributed as Chi-square with one degree offreedom, 12.482; p<0.0001).

Differences between DHEA treated patients and the national IVF databecame even more obvious after age-stratification. Table 9 and FIG. 8summarize age-specific rates in numerical and graphic formats:Miscarriage rates at all ages were lower in DHEA patients than in the2004 national IVF data. Those differences were, however, only after age35 years pronounced.

Here reported data, after DHEA supplementation, demonstrate in womenwith DOR significantly lower miscarriage rates than in a standard IVFcontrol population, a finding particularly pronounced above age 35years. This remarkable observation is further enhanced by the wellrecognized and reported excessive miscarriage risk of women with DOR.Levi et al, for example, reported that women with DOR experiencemiscarriage rates far in excess of standard IVF patients with normalovarian reserve: 57.1 percent under age 35; of 63.5 percent between ages35 and 40 and as high as 90 percent above age 40 years.

Patients in the here reported study that suffered from DOR is bestdocumented by them receiving DHEA supplementation. Under our center'sDHEA protocols, except for women above age 40 years, DHEAsupplementation is offered only to women who have failed at least oneprior IVF cycle with retrieval of less than four oocytes and, therefore,have been designated resistant to ovarian stimulation. Moreover, youngerwomen receive DHEA supplementation only if they also demonstrateelevated age-specific FSH levels. Finally, DHEA supplementation isvoluntary, allowing for the assumption that more severely compromisedpatients, with poorer past IVF experiences, will more likely choosesupplementation.

In contrast, USA IVF outcome data only in a minority represent womenwith diminished ovarian reserve. As Levi et al demonstrated, controlpopulations, therefore, should demonstrate significantly lowermiscarriage rates than our study patients. The finding that women onDHEA supplementation demonstrate in all age groups, but especially aboveage 35, significantly lower miscarriages than the much more favorablenational IVF population is, therefore, noteworthy.

That this difference is less obvious under age 35, only strengthens thevalidity of the here utilized controls. Indeed, the larger degree ofreduction in miscarriage rates in older women should not surprise:Aneuploidy rates increase with age, and age 35 is generally consideredthe cut off, when invasive prenatal genetic prenatal screening becomesindicated. Assuming a beneficial effect of DHEA on aneuploidy rates, alarger effect after age 35 should, therefore, be expected.

Levi et al, reported in women with diminished ovarian reserve above age40 an approximately 90% miscarriage rate. Since older women producefewer embryos, the relative benefits from decreases in aneuploidy rateon number of euploid embryos, transferred into the uterus, will increasewith advancing female age.

Aneuploidy is, however, even in young women a frequent finding. In womenwith diminished ovarian reserve Levi et al reported an almost 60 percentmiscarriage rate under age 35 years. As women physiologically age, theprevalence of aneuploidy continues to increases, reaching close to 90percent in the mid-40ies. Premature ovarian aging, however, does notprematurely enhance aneuploidy rates, and instead maintains expectedage-specific aneuploidy rates. Though demonstrating features of ovarianaging, affected women, therefore, still experience age-appropriateimplantation—and pregnancy rates. Because of decreased oocyte and embryoyields, they, however, do demonstrate reduced cumulative pregnancyrates. Even though significantly affected by prematurely diminishedovarian reserve, a smaller benefit from DHEA under age 35 in our studypopulation should, therefore, not surprise.

By demonstrating in a very high risk population for spontaneouspregnancy loss a statistical association between DHEA supplementationand decreased miscarriage rates, this study does not proof causation.The study, therefore, does not prove that DHEA decreases miscarriage oraneupoidy rates in human embryos. The here reported data, however, offerenough circumstantial evidence to suggest that DHEA may, indeed, exertboth of these effects and, therefore, warrant further investigations. Asuggestion of improved euploidy after DHEA supplementation was, afterall, also observed in human embryos.

Our center's miscarriage rates in women with DOR, prior to introductionof DHEA supplementation, were higher than the national rate seen in thehere utilized control population. The program's pregnancy rates in thesewomen were then only in low single digits. The gradual introduction ofDHEA supplementation between 2004 and 2007 progressively improvedpregnancy rates at our center. Increasing pregnancy numbers anecdotallysuggested a concomitant decline in miscarriage rates. This observation,in turn, lead to the previously noted investigation of aneuploidy ratesin embryos after DHEA supplementation, which, though statisticallyunderpowered, was supportive of a beneficial DHEA effect on ploidy.

The New York center's pregnancy and miscarriage data, alone, were,however, not large enough to allow for statistically valid conclusionsabout factual miscarriage rates. Such conclusions became possible, oncethe independently collected Toronto data became available, andstatistical analysis demonstrated that the two data sets could beunified. At this point the question arose how to control the twocenters' miscarriage experiences. A statistical comparison to a largeand unselected, national data asset appeared appropriate.

While such a comparison cannot replace the gold standard of studydesign, —the prospectively randomized and placebo controlled study, thehere presented data, nevertheless, offer valuable new information. We inthis study used carefully vetted statistical methodologies, which areappropriate for the kind of comparisons offered. Moreover, we evenperformed a second statistical analysis, based on a differentstatistical model, which suggested an even bigger beneficial statisticaleffect of DHEA supplementation, increasing the potential benefit from anapproximately 50 percent to an approximately 80 percent reduction inmiscarriage risk.

Whether the benefit of DHEA supplementation is, indeed, 50 or 80 percentcan as of this moment not be ascertained with certainty, but also shouldnot matter. What seems of importance is the observation that DHEAsupplementation, at least in women with DOR, who characteristicallydemonstrate abnormally high miscarriage rates, appears to significantlyreduce the risk for spontaneous pregnancy loss.

Our here presented data may help to explain why DHEA supplementationincreases egg and embryo quality, improves pregnancy rates and speeds uptime to conception. Egg and embryo quality is, of course, at leastpartially a reflection of ploidy. More euploid embryos will lead to morepregnancies, thus shortening time to conception.

It is important to note that DHEA supplementation, as described, appearssafe and results in only minor side effects. Since DHEA is a mildandrogen but is converted into testosterone (and estradiol), it shouldnot surprise that observed mild side effects, such as oily skin, mildacne vulgaris and hair loss are mostly androgenic in nature.

Embryo selection and improving embryo ploidy have been the rational forattempts at improving pregnancy rates and reducing miscarriage rates viapreimplantation genetic screening (PGS), a concept recently seriouslyquestioned. Here presented data suggest that DHEA supplementation mayresult in more cost effective improvements in ploidy without laboratoryintervention.

Though infertile women with normal ovarian reserve experiencesignificantly lower miscarriage rates than DOR patients, they stillexperience higher miscarriage rates than average populations. Herereported miscarriage rates in DHEA treated DOR patients are, therefore,remarkably low and practically identical to those reported for generalpopulations. Caution should, however, nevertheless, be exercised inconcluding that observed DHEA effect can automatically be extrapolatedto normal, fertile populations, though such a possibility deservesfurther investigation. If confirmed, one could perceive DHEA as aroutine preconception supplement, akin to prenatal vitamins, even inwomen with no fertility problems.

CONCLUSIONS

Based on the hypothesis that major disturbances in chromosome alignmenton the meiotic spindle of oocytes (i.e., congression failure) resultfrom complex interplay of signals, regulating folliculogenesis(increasing the risk of non-disjunction errors), Hodges et al suggestedthat it may be possible to develop prophylactic treatments which canreduce the risk of age-related aneuploidy. This study suggests that DHEAmay, indeed, be a first such drug.

Should efficacy of DHEA supplementation be proven not only in infertilepatients but also in general populations, the potential significance onpublic health could be considerably and by far exceed the more imminentutilization of DHEA in fertility practice.

Example 9

Amidst considerable gains in the treatment of infertility, diminishedovarian reserve (DOR), whether due to physiologic aging of the ovariesor premature ovarian aging (POA), represents one of the few unresolvedproblem of modern infertility care. Indeed, as treatment success withother infertility problems has improved, POA patients increasinglyappear to concentrate in infertility centers, and women above age 40years have become the proportionally most rapidly growing age group inU.S. maternity wards, concomitantly graying the population underinfertility treatments.

Dehydroepiandrosterone (DHEA) supplementation of women with DOR maypositively impact ovarian function by increasing oocyte yields afterstimulation with gonadotropins. We confirmed, and expanded on thisobservation by demonstrating that DHEA also improves egg and embryoquality, pregnancy rates, time to conception and reduces miscarriagerates.

Women with significant degrees of DOR usually have limited time left toconceive with use of autologous oocytes and, as two recently cancelledclinical trials (in the U.S. and Europe) demonstrate, are, therefore,reluctant to enter prospectively randomized studies that may assign themto placebo. All so far published DHEA data are, therefore, either cohortor case controlled studies, representing lower levels of evidence.

In the absence of prospectively randomized, placebo controlled studies,we searched for alternatives. Since DHEA apparently increases oocytesyield, it, likely, positively affects ovarian reserve (OR). OR hastraditionally been investigated utilizing baseline follicle stimulatinghormone (FSH). More recently, anti-Müllerian hormone (AMH) has, however,been suggested as a more specific reflection of OR. Its utilization inassociation with prematurely DOR has been advocated. This study,therefore, utilized AMH to assess OR following DHEA supplementation.

Materials and Methods of Example 9

The study is a cross-sectional analysis of 120 consecutive women withDOR in whom AMH levels were evaluated as a reflection of OR. Theypresented during 2007/8 to our center for initial infertilityconsultation. First AMH levels obtained were used for INITIAL analysis.Post DHEA initiation, exposure to the supplement ranged from 34 to 119days (mean 73±27 days). Women with two or more consecutive AMH levelscomprised patients in the longitudinal study evaluation of OR afterinitiation of DHEA supplementation.

Our center defines DOR in women under age 40 by elevated age-specificFSH levels, as previously reported in detail, or by universal AMH levelsbelow 0.8 ng/ml, which approximately correlate to an FSH of 11.0 mIU/ml.Since OR declines with advancing female age, women above age 40 areuniformly assumed to suffer from DOR. Age-specific FSH levels have inassociation with in vitro fertilization (IVF) been demonstrated todiscriminate between oocytes yields.

FSH and estradiol were evaluated by standard enzyme-linkedimmunoabsorbent assay (ELISA; AIA-600II, Tohso, Tokyo, Japan). Onlyresults in assay range were considered for statistical evaluation. AMHlevels were also obtained by ELISA. In short, the DSL-14400 activeMüllerian Inhibiting Substance/Anti-Müllerian Hormone (MIS/AMH)Enzyme-Linked immunoabsorbent (ELISA) was utilized (Diagnostic SystemsLaboratories, Inc. Webster, Tex. 77598-41217, USA). This is anenzymatically amplified two-site immunoassay, which does not cross reactwith other members of the TGF-.beta. superfamily, including TGF-.beta.1,BMP4 and ACT. Theoretical sensitivity, or minimum detection limit, ascalculated by interpolation of the mean plus two standard deviations(SD) of eight replicates of the 0 ng/ml MIS/AMH Standard, is 0.006ng/ml. Intra-assay coefficient of variation for an overall average AMHconcentration is .ltoreq.20 percent.

Since 2007, DOR patients are at our center, before being advanced intoIVF, for at least two months supplemented with pharmaceutical grade,micronized and pharmacy compounded DHEA at a dosage of 25 mg TID. DHEAis continued throughout all IVF cycles until conception (second,normally rising positive pregnancy test) or until patients discontinuetreatment with autologous oocytes.

The study population was age-stratified under and above age 38 years,and further stratified, based on whether clinical pregnancy had beenachieved or not. Age 38 was chosen as cut off because it has beenreported to represents the beginning of accelerated decline in ovarianreserve.

Data are shown as means±standard deviation (SD) or as raw numbers andpercentages. Data analysis was performed using SPSS windows, version17.0. Demographic and biochemical data were analyzed with paired orunpaired Student's t-test. A generalized linear model was performed toevaluate the interaction of pregnancy status with days of DHEA exposure,adjusted for age at start of treatment.

DHEA utilization at our center was initially approved by the center'sinstitutional review board (IRB) under various study protocols. Afterpublication of a number of studies, the utilization of DHEA was in 2007routinely expanded to all women above age 40 and to younger women withevidence of diminished ovarian reserve. Patients, nevertheless, arestill mandated to sign a DHEA-specific informed consent, which, amongstother facts, advises them that DHEA by prescription is not approved bythe Food and Drug Administration to treat DOR, and is in the UnitedStates commercially available as a food supplement without prescription.

The center's IRB allows for expedited review of studies, which onlyinvolve review of medical records since all patients at initialconsultation sign an informed consent, which allows for such reviews forresearch purposes as long as the medical record remains confidential andthe identity of the patient is protected.

Results of Example 9

The patient population comprised 74% Caucasians, 11% African Americanand 15% Asian patients. A large majority (85%) were recorded with aprimary diagnosis of DOR, 3% with male factor infertility and 12% withtubal factor.

Table 10 below summarizes the characteristics of the study populations,separately for cross-sectional (n=120) and longitudinal assessments(n=55). Obviously, low baseline AMH and high FSH levels are confirmatoryof significant DOR in the study population. Age ranges also confirm thatthe younger patient population, indeed, does reflect relatively younginfertility patients and, therefore, with a considerable prevalence ofPOA, while the older age group in principle represents women above age40 years.

TABLE 10 Characteristics of study patients Cross-sectional LongitudinalStudy Group Study Group All <38 years ≧38 years p-value Number ofpatients 120 55 18 37 Age (years); mean ± SD  39 ± 3.9  39 ± 3.1 34.9 ±3.1  42.1 ± 1.2  <0.001 AMH (ng/ml); Mean ± SD 0.32 ± 0.20 0.22 ± 0.220.20 ± 0.16 0.23 ± 0.17 n.s. Baseline FSH (mIU/ml); Mean ± SD 15.9 ±14.1 15.4 ± 9.1  14.2 ± 8.2  18.0 ± 10.8 n.s. Estradiol (pg/ml) Mean ±SD 60.0 ± 50.0 52.3 ± 28.6 56.1 ± 13.6 53.2 ± 36.6 n.s. Maximal DHEA-S(microg/dL)* Mean ± SD 474 ± 145 476 ± 180 475 ± 224 478 ± 180 n.s.*First value obtained 30 days after initiation of DHEA supplementation

Cross-sectional evaluation of the whole patient population (FIG. 9)demonstrates, unadjusted for age, AMH levels as a function of length ofDHEA supplementation. FIG. 9 very clearly demonstrates a steady increasein AMH over time until 120 days after initiation of DHEA (p=0.002). Age(p=0.007) and length of DHEA supplementation (p=0.019) wereindependently associated with AMH levels. Younger women, under age 38years, demonstrated higher AMH levels from baseline, and proportionallyimproved AMH levels over time after initiation of DHEA more than olderwomen at, or above, 38 years.

Very similar results were obtained in longitudinal evaluation: Here, AMHlevels improved from 0.22±0.22 ng/ml at baseline, before start of DHEA,to 0.35±0.03 ng/ml at highest measured peak, an almost 60 percentimprovement in mean (p=0.0001).

Amongst 55 women who had undergone IVF, by time of analysis, 13 (23.64%)conceived a clinical pregnancy. FIG. 10 demonstrates a comparison of AMHlevels after DHEA supplementation in women who did and did not conceive.As the figure demonstrates, those who conceived demonstrated asignificantly better AMH response, following DHEA supplementation, thanunsuccessful patients, whose AMH response remained flat (interaction ofpregnant versus non-pregnant, Wald Chi Square 11.6; df=1; p=0.001).

Discussion of Example 9

By assessing changes in AMH levels, this study for the first timepresents objective evidence that DHEA supplementation positively affectsdiminished ovarian reserve. In concordance with our prior clinicalobservations, this DHEA effect is visible in younger and older ovaries(FIG. 9), though is more pronounced in younger women with POA.

This study also strongly suggests that observed improvements in OR afterDHEA supplementation lead to better pregnancy rates. As Table 10demonstrates, AMH and FSH levels in the here-utilized study populationare highly confirmatory of a significant degree of DOR.

Indeed, over half of the here-investigated patients consulted with ourcenter for the first time after receiving advice elsewhere todiscontinue fertility attempts with autologous oocytes and proceed intoegg donation.

That in such an adversely selected patient population approximately onein four women still conceived with use of autologous oocytes is, initself, a remarkable accomplishment. It is, however, especiallyremarkable that, as FIG. 10 demonstrates, the ovarian response patternto DHEA is so dramatically different between those women who ended upconceiving and those who did not. While those with future pregnanciesdemonstrated remarkable improvements in AMH levels, unsuccessful womendemonstrated generally no response whatsoever to DHEA. They, thus, forpractical purposes can be seen as a control group: where DHEA does notimprove OR, as indicated by generally flat AMH levels, pregnancy is veryunlikely.

While associations in non-randomized studies always have to be viewedwith caution, here reported results appear convincing. First, theobserved pregnancy rate corresponds well to previously publishedclinical observations at our center, following DHEA supplementation.Improved pregnancy rates following DHEA have since also been reported byinvestigators in Greece and Canada.

While we and others have in the past speculated about possiblemechanisms, why DHEA would improve conception rates in women with DORhas remained unknown. We recently suggested that at least part of DHEA'seffect may be a reduction in oocytes and embryo aneuploidy. This study,however, for the first time offers a more direct and clinicallypractical explanation for DHEA effects in women with DOR.

The concept of OR has been based on a presumed remaining follicular poolwithin ovaries. As this pool shrinks, OR, and with it female fecundity,decline. In the process, the size of immature follicular cohorts,recruited each month, also declines. As cohorts decline in size, smallerand smaller follicle numbers reach gonadotropin sensitivity—the laststage of follicular maturation. As a consequence, follicle numbers andoocytes yield in IVF decline with advancing female age, as does femalefecundity in general.

In fertility practice follicle numbers and oocytes yield are consideredultimate measures of OR. Indeed, AMH is increasingly considered a betterreflection of OR because it better predicts oocytes yield in IVF thanFSH. AMH, a dimeric glycoprotein and a member of the transforming growthfactor (TGF) superfamily, is exclusively produced by granulose cells ofearly developing follicles, from primary to antral follicle stages. AMHis, thus, reflective of small, pre-antral follicles but not of the laterstage follicular pool, better represented by FSH levels. AMH appears tobetter reflect total quantity and, possibly, quality of the remainingfollicular pool, and, therefore, to be a better marker of decliningreproductive age, an observation which potentially explains how DHEAaffects ovarian function.

By demonstrating improving AMH levels, this study suggests that inselected patients with DOR, DHEA progressively improves OR at follicularstages at which AMH is produced. This means that over time DHEAincreases the pool of follicles up to pre-antral stage, in this studycausing a steady improvement in AMH up to 120 days post DHEA initiation.In prior clinical studies, with longer follow up periods, wedemonstrated that follicular numbers and oocytes yield increase up toapproximately five months of DHEA supplementation, equal to theapproximate time period from primordial stages to gonadotropinsensitivity.

Combined, these observations suggest two possible mechanisms by whichDHEA exerts its effects, both reflective of impacts on the follicularmaturation cycle and improvements in number of AMH producing follicles:DHEA either positively affects recruitment from the dormant follicularpool or it progressively reduces apoptosis of originally recruitedfollicles, which represents the primary process by which originallyrecruited follicles are eliminated during follicular maturation. Eitherway, progressively more pre-antral follicles accumulate, resulting inthe here documented increase in AMH over time from initiation of DHEAsupplementation.

The effect of DHEA on follicular recruitment has not been investigated.Androgens, in general, appear, however, capable of positively affectingfollicular recruitment in the mouse.

Similarly, nothing is known about DHEA effects on apoptosis, andandrogens, in general, have been reported to have both enhancing andsuppressing effects on ovarian granulose cell apoptosis.

Our previously published clinical observations suggested thatapproximately two months of DHEA supplementation were required beforestatistically significant differences in outcomes could be observed.FIG. 10 suggests that beneficial effects of DHEA may already becomeapparent even earlier, and may be reflected in spontaneous pregnancieswe and others have reported in a small number of prognostically highlyunfavorable patients, preceding other therapeutic interventions.

While improving AMH levels in women with DOR appear closely associatedwith pregnancy success, AMH is, unfortunately, not sensitive enough topredict who will or will not conceive.

Pregnancies can even be established at undetectable AMH levels. Thismeans that AMH levels alone will not allow discrimination between whodoes and does not deserve further infertility treatments.

As this study, however, demonstrates, AMH offers objective evidence forthe therapeutic efficacy of DHEA in women with DOR, and especially underage 38 years. Moreover, a good AMH response to DHEA supplementationclearly discriminates between good and poor prognosis patients inregards to pregnancy success. This information alone will greatlyimprove patient counseling in women with significant DOR. We arecurrently investigating other markers of OR in attempts to even betterpredict success of DHEA supplementation and, thus, avoid such treatmentin women who will not improve pregnancy chances in response to DHEAsupplementation.

While the foregoing written description of the invention enables one ofordinary skill to make and use what is considered presently to be thebest mode thereof, those of ordinary skill will understand andappreciate the existence of variations, combinations, and equivalents ofthe specific exemplary embodiments thereof. The invention is thereforeto be limited not by the exemplary embodiments herein, but by allembodiments within the scope and spirit of the appended claims.

1. A method of improving ovarian reserve, comprising: evaluating a firstanti-Müllerian hormone level of a human female; increasing an amount ofpre-antral follicles in the human female's ovarian reserve byadministering an androgen to the female for at least about one month,without requiring concurrent administration of gonadotropin; evaluatinga second anti-Müllerian hormone level of the female; and determiningwhether the number of pre-antral follicles in the ovarian reserveincreased by evaluating whether the second anti-Müllerian hormone levelis greater than the first anti-Müllerian hormone level.
 2. The method ofclaim 1, wherein the human female is under 38 years of age.
 3. Themethod of claim 1, wherein the human female is 38 years of age or older.4. The method of claim 1, wherein the androgen is selected from thegroup consisting of dehydroepiandrosterone, dehydroepiandrosteronesulfate, testosterone and androstenedione.
 5. The method of claim 1,wherein the first anti-Müllerian hormone level of the human female isbelow about 0.08 ng/ml.
 6. The method of claim 1, wherein the humanfemale has a follicle stimulating hormone level below about 11.0 mIU/ml.7. The method of claim 1, wherein the androgen is administered to thefemale until the second anti-Müllerian hormone level is greater than thefirst anti-Müllerian hormone level by a predetermined percentage oramount.
 8. The method of claim 1, wherein the androgen is administeredto the female until the second anti-Müllerian hormone level isapproximately 60 percent greater than the first anti-Müllerian hormonelevel.
 9. The method of claim 1, wherein the first anti-Müllerianhormone level is about 0.22+/−0.22 ng/ml and the second anti-Müllerianhormone level is about 0.35+/−0.03 ng/ml.
 10. The method of claim 1,wherein the at least about one month is about 30 days.
 11. The method ofclaim 1, wherein the at least about one month is between 30 days and 120days.
 12. The method of claim 1, wherein the androgen is administered tothe female for about four months.
 13. The method of claim 1, wherein thestep of evaluating the first or second anti-Müllerian hormone levelcomprises using a standard enzyme-linked immunoabsorbent assay.
 14. Themethod of claim 1, wherein between 50 mg and 100 mg per day of theandrogen is administered to the female.
 15. The method of claim 1,wherein about 75 mg per day of the androgen is administered to thefemale.
 16. The method of claim 1, wherein between 15 mg and 40 mg ofthe androgen is administered three times a day to the female.
 17. Themethod of claim 1, wherein the androgen is orally administered.
 18. Amethod of evaluating the effect of administering an androgen to a humanfemale with diminished ovarian reserve, the method comprising:evaluating a first anti-Müllerian hormone level of the female;administering the androgen to the female for at least about one month,without requiring concurrent administration of gonadotropin, to increasethe amount of pre-antral follicles in the female's ovarian reserve;evaluating a second anti-Müllerian hormone level of the female, andcomparing the second anti-Müllerian hormone level to the firstanti-Müllerian hormone level.
 19. The method of claim 18, wherein theandrogen is administered to the female for about four months.
 20. Themethod of claim 18, wherein the androgen is selected from the groupconsisting of dehydroepiandrosterone, dehydroepiandrosterone sulfate,testosterone and androstenedione.